Genetically modified rat models for severe combined immunodeficiency (scid)

ABSTRACT

This invention relates to the engineering of animal cells, preferably mammalian, more preferably rat, that are deficient due to the disruption of tumor suppressor gene(s) or gene product(s). In another aspect, the invention relates to genetically modified rats, as well as the descendants and ancestors of such animals, which are animal models of human cancer and methods of their use.

BACKGROUND OF THE INVENTION

Gene modification is a process whereby a specific gene, or a fragment ofthat gene, is altered. This alteration of the targeted gene may resultin a change in the level of RNA and/or protein that is encoded by thatgene, or the alteration may result in the targeted gene encoding adifferent RNA or protein than the untargeted gene. The modified gene maybe studied in the context of a cell, or, more preferably, in the contextof a genetically modified animal.

Genetically modified animals are among the most useful research tools inthe biological sciences. An example of a genetically modified animal isa transgenic animal, which has a heterologous (i.e., foreign) gene, orgene fragment, incorporated into their genome that is passed on to theiroffspring. Although there are several methods of producing geneticallymodified animals, the most widely used is microinjection of DNA intosingle cell embryos. These embryos are then transferred intopseudopregnant recipient foster mothers. The offspring are then screenedfor the presence of the new gene, or gene fragment. Potentialapplications for genetically modified animals include discovering thegenetic basis of human and animal diseases, generating diseaseresistance in humans and animals, gene therapy, toxicology studies, drugtesting, and production of improved agricultural livestock.

Identification of novel genes and characterization of their functionusing mutagenesis has also been shown to be productive in identifyingnew drugs and drug targets. Creating in vitro cellular models thatexhibit phenotypes that are clinically relevant provides a valuablesubstrate for drug target identification and screening for compoundsthat modulate not only the phenotype but also the target(s) thatcontrols the phenotype. Modulation of such a target can provideinformation that validates the target as important for therapeuticintervention in a clinical disorder when such modulation of the targetserves to modulate a clinically relevant phenotype.

Animal models exhibiting Severe Combined Immunodeficiency (SCID) haveand important advantage over animals with more limitedimmunodeficiencies due to the lack of B-cells, T-cells and NK-cells.SCID animals readily accept xenografts and, are therefore, a crucialmodel for cancer research. The SCID animal can receive grafts and othertissue transplants (e.g., lymphocytes or tumor cells) without elicitingan immune response. SCID animal models are used for xenotransplantationof cell lines such as cultured human cancer cell lines or cells fromsurgically resected tumors. For example, fragments of tumors resectedfrom patients can transplanted into an anesthetized SCID mouse. Thexenografts are stable (i.e. not rejected by the host's immune system),and are useful for a wide range of studies. The histological studies ofsuch xenografts show that they maintain major features such as cysts,and mono-or-multilocular cavities, as the original tumors. The SCIDanimal xenografts are therefore relevant for human tumor biologystudies. SCID animal xenografts are also useful for examination of knowncancer genes such as tumor suppressor genes and the potential discoveryof new cancer targets. In one method, tumor tissues from the SCID animalxenografts are taken, RNA is extracted, reverse transcribed, and PCRamplified. The analysis of sequences can identify mutations in genesthat are associated with the tumor or cancer. In another method, afunctional assay can he performed to identify genes that may be over orunder expressed in the tumor. The SCID animal xenografts are also usefulfor studies of the efficacy of potential oncoceuticals. In one method, aradiological growth assay is employed to determine tumor growth delay.The tumor is typically given a single dose of irradiation treatment, andthe tumor size is scored to calculate the growth delay. In a similarfashion, therapeutic agents such as compounds and biologics can betested for tumor or metastasis suppression.

Since the SCID model lacks peripheral B- and T-cell activity, the animalaccepts human grafts including human lymphoid tissues. This techniquegives a SCID model that is reconstituted with human immune systemcomponents. One example is the engraftment of human peripheral bloodlymphocytes (PBLs), thymus, and liver tissue into a SCID mouse. The SCIDmodel can therefore mimic human immune response better than a wild typeanimal before actual human clinical trials.

Having the capability of generating rats with humanized organs usingprimary cells either from healthy patients or from patients with geneticlesions associated with various disease states would provide unique andvaluable resources for drug discovery and therapeutic research programs.The organs include, but are not limited to, liver, pancreas, skin andintestine. Since the SCID rats lack peripheral T-cell and NK-cellactivity, transplanted human cells will not be rejected and willincorporate into the tissue at the site of injection. The rat will thencontain an organ which consists of significant numbers of human cells.In the best case scenario, the organ will completely be composed ofhuman cells, although organs with lower percentages of human cellsremain useful for drug discovery and therapeutic research

In some applications it may be more desirable to generate rat organswith lower levels of human cells (chimeric organs) using primary cellseither from healthy patients or from patients with genetic lesionsassociated with various disease states. These models would provideunique and valuable resources for drug discovery and therapeuticresearch programs. Such examples include, but are not limited to brain,heart and skeletal muscle, where it may not be possible to generatecompletely humanized organs.

Animal models which exhibit the SCID phenotype are used for immunologicstudies. These experiments usually involve but are not limited to thetesting for the presence and measurement of leukocyte populations, andthe functionality of immunocompetent cells. In order to test for thepresence of immune cells, flow cytometry can be done using cellssuspensions from the thymuses and spleen. Antibodies can target immunecell surface antigens such as Cd3, and identify leukocytes, whichcontain B-, T-, and NK-cell populations. Cell numbers can be compared tocontrol populations to indicate whether the immune system has beencompromised. To test the functionality of immune cells, one may test theproliferation of spleen cells when stimulated by B- and T-cell mitogens.The mitogens will stimulate cell growth and division in functionalimmune cells. The amount of proliferation reflects the functionality ofimmunocompetent cells.

SCID animal models are very useful to test immune response to infectiousdiseases and pathogens, such as but not limited to Mycobacteriumtuberculosis. Recurrent M. tuberculosis and other infections frequentlyoccur in both SCID patients and SCID animal models. Many experiments canbe employed to measure the progression of infectious diseases andalleviation of the disease due to therapeutic intervention. To measurethe virulence of bacteria in infectious disease studies, SCID animalmodels can be infected via aerosol inhalation. Lung cells and tissue arethen collected and plated on agar to count the number of colony formingunits (CFU). The amount of CFU produced over time is an indicator ofdisease progression. Residential alveolar macrophages play a substantialrole in protection against infectious disease; therefore, cytokineassays can be done to determine what cell types are recruited to thealveolar space during disease progression. Bronchoalveolar lavage cells(BAL) are harvested from the trachea, put on a slide and stained for thepresence of cytokines such as Ccl2. The numbers and types of cytokinespresent are known to play different roles and can be measured to monitordisease progression.

The SCID rat, as compared to other SCID models, is particularly usefulfor many applications, including but not limited to drug testing,toxicology models, humanized organ production, immunologic, andinfectious disease models. The SCID rat has many advantages over theSCID mouse model. The rat is has been known to be a better animal modelfor many human disease states for over 50 years. The rat performs mostmajor medical assays with a higher proficiency than mice. The size ofthe rat is also important. Study by instrumentation, nerve conduction,surgery, and imaging are all more efficient in the rat. Blood, tissue,and tumor sampling are all easier and more accurate in the rat. The ratalso provides up to ten times more tissue for more conclusive data.

SCID models will provide an immunocompromised model that can serve as arecipient of transplanted stem cells or in vitro differentiated stemcells. Examples of stem cells include, but are not limited to,embryonic, amniotic, umbilical cord-, mesenchymal -, hepatic- or adiposestromal, induced pluripotent cell-derived populations. Stem cells ordifferentiated stem cells obtained from healthy or diseased patients canbe used to produce organs comprised entirely of human cells (humanizedorgans) or organs with significant percentages of human cells (chimericorgans).

Animal models exhibiting clinically relevant phenotypes are alsovaluable for drug discovery and development and for drug targetidentification. For example, mutation of somatic or germ cellsfacilitates the production of genetically modified offspring or clonedanimals having a phenotype of interest. Such animals have a number ofuses, for example as models of physiological disorders (e.g., of humangenetic diseases) that are useful for screening the efficacy ofcandidate therapeutic compounds or compositions for treating orpreventing such physiological disorders. Furthermore, identifying thegene(s) responsible for the phenotype provides potential drug targetsfor modulating the phenotype and, when the phenotype is clinicallyrelevant, for therapeutic intervention. In addition, the manipulation ofthe genetic makeup of organisms and the identification of new genes haveimportant uses in agriculture, for example in the development of newstrains of animals and plants having higher nutritional value orincreased resistance to environmental stresses (such as heat, drought,or pests) relative to their wild-type or non-mutant counterparts.

Since most eukaryotic cells are diploid, two copies of most genes arepresent in each cell. As a consequence, mutating both alleles to createa homozygous mutant animal is often required to produce a desiredphenotype, since mutating one copy of a gene may not produce asufficient change in the level of gene expression or activity of thegene product from that in the non-mutated or wild-type cell ormulticellular organism, and since the remaining wild-type copy wouldstill be expressed to produce functional gene product at sufficientlevels. Thus, to create a desired change in the level of gene expressionand/or function in a cell or multicellular organism, at least twomutations, one in each copy of the gene, are often required in the samecell.

In other instances, mutation in multiple different genes may be requiredto produce a desired phenotype. In some instances, a mutation in bothcopies of a single gene will not be sufficient to create the desiredphysiological effects on the cell or multi-cellular organism. However, amutation in a second gene, even in only one copy of that second gene,can reduce gene expression levels of the second gene to produce acumulative phenotypic effect in combination with the first mutation,especially if the second gene is in the same general biological pathwayas the first gene. This effect can alter the function of a cell ormulti-cellular organism. A hypomorphic mutation in either gene alonecould result in protein levels that are severely reduced but with noovert effect on physiology. Severe reductions in the level of expressionof both genes, however, can have a major impact. This principle can beextended to other instances where mutations in multiple (two, three,four, or more, for example) genes are required cumulatively to producean effect on activity of a gene product or on another phenotype in acell or multi-cellular organism. It should be noted that, in thisinstance, such genes may all be expressed in the same cell type andtherefore, all of the required mutations occur in the same cell.However, the genes may normally be expressed in different cell types(for example, secreting the different gene products from the differentcells). In this case, the gene products are expressed in different cellsbut still have a biochemical relationship such that one or moremutations in each gene is required to produce the desired phenotype.

BRIEF SUMMARY OF THE INVENTION

In accordance with the purposes of this invention, as embodied andbroadly described herein, this invention relates to the engineering ofanimal cells, preferably mammalian, more preferably rat, that aredeficient due to the disruption of gene(s) or gene product(s) resultingin Severe Combined Immunodeficiency (SCID).

In another aspect, the invention relates to genetically modified rats,as well as the descendants and ancestors of such animals, which areanimal models of human SCID and methods of their use.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWING

This invention, as defined in the claims, can be better understood withreference to the following drawings:

FIGS. 1-4 show the process for creating a genetically modified SCID ratmodel using DNA transposons to create an insertion mutation directly inthe germ line.

FIG. 1: Gene modification by DNA transposons.

FIG. 2: Breeding strategy for creating rat knockouts directly in thegerm cells with DNA transposons.

FIG. 3: DNA sequences.

FIG. 4: DNA transposon-mediated insertion mutation in Rattus norvegicusAda gene.

In the following description of the illustrated embodiments, referencesare made to the accompanying drawings, which form a part hereof, and inwhich is shown by way of illustration various embodiments in which theinvention may be practiced. It is to be understood that otherembodiments may be utilized and structural and functional changes may bemade without departing from the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods, devices, andmaterials are now described. All references, publications, patents,patent applications, and commercial materials mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the materials and/or methodologies which are reported in thepublications which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that thisinvention is not limited to specific synthetic methods, specificrecombinant biotechnology methods unless otherwise specified, or toparticular reagents unless otherwise specified, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only and is notintended to he limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

A “coding sequence” or a sequence “encoding” an expression product, suchas a RNA, polypeptide, protein, or enzyme, is a nucleotide sequencethat, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

“Complementary,” as used herein, refers to the subunit sequencecomplementarity between two nucleic acids, e.g., two DNA molecules. Whena nucleotide position in both of the molecules is occupied bynucleotides normally capable of base pairing with each other, then thenucleic acids are considered to be complementary to each other at thisposition. Thus, two nucleic acids are complementary to each other when asubstantial number (at least 50%) of corresponding positions in each ofthe molecules are occupied by nucleotides which normally base pair witheach other (e.g., A:T and G:C nucleotide pairs).

A “deletion mutation” means a type of mutation that involves the loss ofgenetic material, which may be from a single base to an entire piece ofchromosome. Deletion of one or more nucleotides in the DNA could alterthe reading frame of the gene; hence, it could result in a synthesis ofa nonfunctional protein due to the incorrect sequence of amino acidsduring translation.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g., theresulting protein, may also be said to be “expressed”. An expressionproduct can be characterized as intracellular, extracellular orsecreted. The term “intracellular” means something that is inside acell. The term “extracellular” means something that is outside a cell. Asubstance is “secreted” by a cell if it appears in significant measureoutside the cell, from somewhere on or inside the cell.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include introns and regulatory DNA sequences, such aspromoter sequences, 5′-untranslated region, or 3′-untranslated regionwhich affect for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription.

By “genetically modified” is meant a gene that is altered from itsnative state (e.g., by insertion mutation, deletion mutation, nucleicacid sequence mutation, or other mutation), or that a gene product isaltered from its natural state (e.g., by delivery of a transgene thatworks in trans on a gene's encoded mRNA or protein, such as delivery ofinhibitory RNA or delivery of a dominant negative transgene).

By “exon” is meant a region of a gene which includes sequences which areused to encode the amino acid sequence of the gene product.

The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.

Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature.

As used herein, the term “homology” refers to the subunit sequenceidentity or similarity between two polymeric molecules e.g., between twonucleic acid molecules, e.g., between two DNA molecules, or twopolypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two polypeptide molecules is occupied by phenylalanine, thenthey are identical at that position. The homology between two sequences,most clearly defined as the % identity, is a direct function of thenumber of identical positions, e.g., if half (e.g., 5 positions in apolymer 10 subunits in length) of the positions in two polypeptidesequences are identical then the two sequences are 50% identical; if 70%of the positions, e.g., 7 out of 10, are matched or homologous, the twosequences share 70% identity. By way of example, the polypeptidesequences ACDEFG and ACDHIK share 50% identity and the nucleotidesequences CAATCG and CAAGAC share 50% identity. It should be understoodthat a sequence identity of at least 90%, such as 90% identity, 91%identity, 92% identity, 93% identity, 94% identity, 95% identity, 96%identity, 97% identity, 98% identity, or 99% identity applies to allsequences disclosed in the present application.

“Homologous recombination” is the physical exchange of DNA expedited bythe breakage and reunion of two non-sister chromatids. In order toundergo recombination the DNA duplexes must have complimentarity. Themolecular mechanism is as follows: DNA duplexes pair, homologous strandsare nicked, and broken strands exchange DNA between duplexes. The regionat the site of recombination is called the hybrid DNA or heteroduplexDNA. Second nicks are made in the other strand, and the second strandcrosses over between duplexes. After this second crossover event thereciprocal recombinant or splice recombinant is created. The duplex ofone DNA parent is covalently linked to the duplex of another DNA parent.Homologous recombination creates a stretch of heteroduplex DNA.

A “hypomorphic mutation” is a change to the genetic material (usuallyDNA or RNA), which can be caused by any form of genetic mutation, andcauses an decrease in normal gene function without causing a completeabsence of normal gene function.

The term “inbred animal” is used herein to refer to an animal that hasbeen interbred with other similar animals of the same species in orderto preserve and fix certain characteristics, or to prevent othercharacteristics from being introduced into the breeding population.

The term “insertional mutation” is used herein to refer thetranslocation of nucleic acid from one location to another locationwhich is in the genome of an animal so that it is integrated into thegenome, thereby creating a mutation in the genome. Insertional mutationscan also include knocking out or knocking in of endogenous or exogenousDNA via gene trap or cassette insertion. Exogenous DNA can access thecell via electroporation or chemical transformation. If the exogenousDNA has homology with chromosomal DNA it will align itself withendogenous DNA. The exogenous DNA is then inserted or disrupts theendogenous DNA via two adjacent crossing over events, known ashomologous recombination. A targeting vector can use homologousrecombination for insertional mutagenesis. Insertional mutagenesis ofendogenous or exogenous DNA can also be carried out via DNA transposon.The DNA transposon is a mobile element that can insert itself along withadditional exogenous DNA into the genome. Insertional mutagenesis ofendogenous or exogenous DNA can be carried out by retroviruses.Retroviruses have a RNA viral genome that is converted into DNA byreverse transcriptase in the cytoplasm of the infected cell. Linearretroviral DNA is transported into the nucleus, and become integrated byan enzyme called integrase. Insertional mutagenesis of endogenous orexogenous DNA can also be done by retrotransposons in which an RNAintermediate is translated into DNA by reverse transcriptase, and theninserted into the genome.

The term “gene knockdown” refers to techniques by which the expressionof one or more genes is reduced, either through genetic modification (achange in the DNA of one of the organism's chromosomes) or by treatmentwith a reagent such as a short DNA or RNA oligonucleotide with asequence complementary to either an mRNA transcript or a gene. Ifgenetic modification of DNA is done, the result is a “knockdownorganism” or “knockdowns”.

By “knock-out” is meant an alteration in the nucleic acid sequence thatreduces the biological activity of the polypeptide normally encodedtherefrom by at least 80% compared to the unaltered gene. The alterationmay be an insertion, deletion, frameshift mutation, or missensemutation. Preferably, the alteration is an insertion or deletion, or isa frameshift mutation that creates a stop codon.

An “L1 sequence” or “L1 insertion sequence” as used herein, refers to asequence of DNA comprising an L1 element comprising a 5′ UTR, ORF1 andORF2, a 3′ UTR and a poly A signal, wherein the 3′ UTR has DNA (e.g., agene trap or other cassette) positioned either therein or positionedbetween the 3′ UTR and the poly A signal, which DNA is to be insertedinto the genome of a cell.

A “mutation” is a detectable change in the genetic material in theanimal, which is transmitted to the animal's progeny. A mutation isusually a change in one or more deoxyribonucleotides, the modificationbeing obtained by, for example, adding, deleting, inverting, orsubstituting nucleotides. Exemplary mutations include but are notlimited to a deletion mutation, an insertion mutation, a non-sensemutation or a missense mutation. Thus, the terms “mutation” or “mutated”as used herein are intended to denote an alteration in the “normal” or“wild-type” nucleotide sequence of any nucleotide sequence or region ofthe allele. As used herein, the terms “normal” and “wild-type” areintended to be synonymous, and to denote any nucleotide sequencetypically found in nature. The terms “mutated” and “normal” are thusdefined relative to one another; where a cell has two chromosomalalleles of a gene that differ in nucleotide sequence, at least one ofthese alleles is a “mutant” allele as that term is used herein. Based onthese definitions, an “endogenous SCID gene” is the “wild-type” genethat exists normally in a cell, and a “mutated SCID gene” defines a genethat differs in nucleotide sequence from the wild-type gene.

“Non-homologous end joining (NHEJ)” is a cellular repair mechanism. TheNHEJ pathway is defined by the ligation of blunt ended double stand DNAbreaks. The pathway is initiated by double strand breaks in the DNA, andworks through the ligation of DNA duplex blunt ends. The first step isrecognition of double strand breaks and formation of scaffold. Thetrimming, filling in of single stranded overhangs to create blunt endsand joining is executed by the NHEJ pathway. An example of NHEJ isrepair of a DNA cleavage site created by a zinc finger nuclease (ZFN).This would normally be expected to create a small deletion mutation.

“Nucleic Acid sequence mutation” is a mutation to the DNA of a gene thatinvolves change of one or multiple nucleotides. A point mutation whichaffects a single nucleotide can result in a transition (purine to purineor pyrimidine to pyrimidine) or a transversion (purine to pyrimidine orpyrimidine to purine). A point mutation that changes a codon torepresent a different amino acid is a missense mutation. Some pointmutations can cause a change in amino acid so that there is a prematurestop codon; these mutations are called nonsense mutations. A mutationthat inserts or deletes a single base will change the entire downstreamsequence and are known as frameshift mutations. Some mutations change abase pair but have no effect on amino acid representation; these arecalled silent mutations. Mutations to the nucleic acid of a gene canhave different consequences based on their location (intron, exon,regulatory sequence, and splice joint).

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

The term “outbred animal” is used herein to refer to an animal thatbreeds with any other animal of the same species without regard to thepreservation of certain characteristics.

As used herein, the term “phenotype” means any property of a cell ororganism. A phenotype can simply he a change in expression of an mRNA orprotein. Examples of phenotypes also include, but are in no way limitedto, cellular, biochemical, histological, behavioral, or whole organismalproperties that can be detected by the artisan. Phenotypes include, butare not limited to, cellular transformation, cell migration, cellmorphology, cell activation, resistance or sensitivity to drugs orchemicals, resistance or sensitivity to pathogenic protein localizationwithin the cell (e.g., translocation of a protein from the cytoplasm tothe nucleus), resistance or sensitivity to ionizing radiation, profileof secreted or cell surface proteins, (e.g., bacterial or viral)infection, post-translational modifications, protein localization withinthe cell (e.g., translocation of a protein from the cytoplasm to thenucleus), profile of secreted or cell surface proteins, cellproliferation, signal transduction, metabolic defects or enhancements,transcriptional activity, recombination intermediate joining, DNA damageresponse, cell or organ transcript profiles (e.g., as detected usinggene chips), apoptosis resistance or sensitivity, animal behavior, organhistology, blood chemistry, biochemical activities, gross morphologicalproperties, life span, tumor susceptibility, weight, height/length,immune function, organ function, any disease state, and other propertiesknown in the art. In certain situations and therefore in certainembodiments of the invention, the effects of mutation of one or moregenes in a cell or organism can be determined by observing a change inone or more given phenotypes (e.g., in one or more given structural orfunctional features such as one or more of the phenotypes indicatedabove) of the mutated cell or organism compared to the same structuralor functional feature(s) in a corresponding wild-type or (non-mutated)cell or organism (e.g., a cell or organism in which the gene(s) have notbeen mutated).

By “plasmid” is meant a circular strand of nucleic acid capable ofautosomal replication in plasmid-carrying bacteria. The term includesnucleic acid which may be either DNA or RNA and may be single- ordouble-stranded. The plasmid of the definition may also include thesequences which correspond to a bacterial origin of replication.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. The promoter may be operatively associated with otherexpression control sequences, including enhancer and repressorsequences.

A “random site” is used herein to refer to a location in the genomewhere a retrotransposition or transposition or other DNA mutation eventtakes places, without prior intention of mutation at that particularlocation. It is also used herein to refer to a location in the genomethat is randomly modified by any insertion mutation or deletion mutationor nucleic acid sequence mutation.

The term “regulatory sequence” is defined herein as including promoters,enhancers and other expression control elements such as polyadenylationsequences, matrix attachment sites, insulator regions for expression ofmultiple genes on a single construct, ribosome entry/attachment sites,introns that are able to enhance expression, and silencers.

By “reporter gene” is meant any gene which encodes a product whoseexpression is detectable. A reporter gene product may have one of thefollowing attributes, without restriction: fluorescence (e.g., greenfluorescent protein), enzymatic activity (e.g., 1acZ or luciferase), oran ability to be specifically bound by a second molecule (e.g., biotinor an antibody-recognizable epitope).

By “retrotransposition” as used herein, is meant the process ofintegration of a sequence into a genome, expression of that sequence inthe genome, reverse transcription of the integrated sequence to generatean extrachromosomal copy of the sequence and reintegration of thesequence into the genome.

A “retrotransposition event” is used herein to refer to thetranslocation of a retrotransposon from a first location to a secondlocation with the preferable outcome being integration of aretrotransposon into the genome at the second location. The processinvolves a RNA intermediate, and can retrotranspose from one chromosomallocation to another or from introduced exogenous DNA to endogenouschromosomal DNA.

By “selectable marker” is meant a gene product which may be selected foror against using chemical compounds, especially drugs. Selectablemarkers often are enzymes with an ability to metabolize the toxic drugsinto non-lethal products. For example, the pac (puromycin acetyltransferase) gene product can metabolize puromycin, the dhfr geneproduct can metabolize trimethoprim (tmp) and the bla gene product canmetabolize ampicillin (amp). Selectable markers may convert a benigndrug into a toxin. For example, the HSV tk gene product can change itssubstrate, FIAU, into a lethal substance. Another selectable marker isone which may be utilized in both prokaryotic and eukaryotic cells. Theneo gene, for example, metabolizes and neutralizes the toxic effects ofthe prokaryotic drug, kanamycin, as well as the eukaryotic drug, G418.

By “selectable marker gene” as used herein is meant a gene or otherexpression cassette which encodes a protein which facilitatesidentification of cells into which the selectable marker gene isinserted.

A “specific site” is used herein to refer to a location in the genomethat is predetermined as the position where a retrotransposition ortransposition event or other DNA mutation will take place. It is alsoused herein to refer to a specific location in the genome that ismodified by any insertion mutation or deletion mutation or nucleic acidsequence mutation.

A “SCID gene” is used herein to refer to a gene which encodes a proteinthat is associated with the phenotype that is characterized as SevereCombined Immunodeficiency (SCID). This phenotype ranges from early onset(infancy), delayed, and late-onset (adulthood). The phenotype may alsovary in the extent of immune insufficiency from severe infections togradual immunologic debilitation. The phenotype encompasses all B-, T-,and NK-cell deficiencies and combinations of individual celldeficiencies. A “SCID protein” is used herein to refer to a proteinproduct of a gene that is associated with the phenotype that ischaracterized as SCID.

As used herein, the term “targeted genetic recombination” refers to aprocess wherein recombination occurs within a DNA target locus presentin a host cell or host organism. Recombination can involve eitherhomologous or non-homologous DNA.

The term “transfection” means the introduction of a foreign nucleic acidinto a cell. The term “transformation” means the introduction of a“foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence toan ES cell or pronucleus, so that the cell will express the introducedgene or sequence to produce a desired substance in a geneticallymodified animal.

By “transgenic” is meant any animal which includes a nucleic acidsequence which is inserted by artifice into a cell and becomes a part ofthe genome of the animal that develops from that cell. Such a transgenemay be partly or entirely heterologous to the transgenic animal.Although transgenic mice represent another embodiment of the invention,other transgenic mammals including, without limitation, transgenicrodents (for example, hamsters, guinea pigs, rabbits, and rats), andtransgenic pigs, cattle, sheep, and goats are included in thedefinition.

By “transposition” as used herein, is meant the process of one DNAsequence insertion into another (location) without relying on sequencehomology. The DNA element can be transposed from one chromosomallocation to another or from introduction of exogenous DNA and insertedinto the genome.

A “transposition event” or “transposon insertion sequence” is usedherein to refer to the translocation of a DNA transposon either from onelocation on the chromosomal DNA to another or from one location onintroduced exogenous DNA to another on the chromosomal DNA.

By “transposon” or “transposable element” is meant a linear strand ofDNA capable of integrating into a second strand of DNA which may helinear or may be a circularized plasmid. Transposons often have targetsite duplications, or remnants thereof, at their extremities, and areable to integrate into similar DNA sites selected at random, or nearlyrandom. Preferred transposons have a short (e.g., less than 300) basepair repeat at either end of the linear DNA. By “transposable elements”is meant any genetic construct including but not limited to any gene,gene fragment, or nucleic acid that can be integrated into a target DNAsequence under control of an integrating enzyme, often called atransposase.

A coding sequence is “under the control of” or “operatively associatedwith” transcriptional and translational control sequences in a cell whenRNA polymerase transcribes the coding sequence into mRNA, which is thentrans-RNA spliced (if it contains introns) and translated, in the caseof mRNA, into the protein encoded by the coding sequence.

The term “variant” may also be used to indicate a modified or alteredgene, DNA sequence, enzyme, cell, etc., i.e., any kind of mutant.

The term “vector” is used interchangeably with the terms “construct”,“cloning vector” and “expression vector” and means the vehicle by whicha DNA or RNA sequence (e.g., a foreign gene) can be introduced into ahost cell, (e.g., ES cell or pronucleus) so as to transform the host andpromote expression (e.g., transcription and translation) of theintroduced sequence including but not limited to plasmid, phage,transposons, retrotransposons, viral vector, and retroviral vector. By“non-viral vector” is meant any vector that does not comprise a virus orretrovirus.

A “vector sequence” as used herein, refers to a sequence of DNAcomprising at least one origin of DNA replication and at least oneselectable marker gene.

For the purposes of the present invention, the term “zinc fingernuclease” or “ZFN” refers to a chimeric protein molecule comprising atleast one zinc finger DNA binding domain effectively linked to at leastone nuclease or part of a nuclease capable of cleaving DNA when fullyassembled. Ordinarily, cleavage by a ZFN at a target locus results in adouble stranded break (DSB) at that locus.

The present invention provides a desired rat or a rat cell whichcontains a predefined, specific and desired alteration rendering the rator rat cell predisposed to Severe Combined Immunodeficiency (SCID) andits variations (autosomal recessive, ionizing radiation sensitive,X-linked, microcephaly, growth retardation). Specifically, the inventionpertains to a genetically altered rat, or a rat cell in culture, that isdefective in at least one of two alleles of a SCID gene such as the Adagene, the Rag1 gene, etc. In one embodiment, the SCID gene is the Adagene. In another embodiment, the SCID gene is one of several known SCIDgenes, such as (Rag1, Rag2, Dclre1e, Nhej1, Jak3, Il7r, Ptprc, Cd3d,Cd3e, Il2rg, Prkdc Sirpa, Foxn1). The inactivation of at least one ofthese SCID alleles results in an animal with a higher susceptibility toSevere Combined Immunodeficiency (SCID) induction. In one embodiment,the genetically altered animal is a rat of this type and is able toserve as a useful model for SCID and as a test animal for oncology andother studies. The invention additionally pertains to the use of suchrats or rat cells, and their progeny in research and medicine.

In one embodiment, the invention provides a genetically modified orchimeric rat cell whose genome comprises two chromosomal alleles of aSCID gene (especially, the Ada gene), wherein at least one of the twoalleles contains a mutation, or the progeny of this cell. The inventionincludes the embodiment of the above animal cell, wherein one of thealleles expresses a normal SCID gene product. The invention includes theembodiment wherein the rat cell is a pluripotent cell such as anembryonic cell, embryonic stem (ES) cell, induced pluripotent stem cell(iPS), or spermatagonial stem (SS) cell, and in particular, wherein theSCID gene is the gene. In another embodiment, the SCID gene is one ofseveral known SCID genes, such as (Rag1, Rag2, Dclre1c, Nhej1, Jak3,Il7r, Ptprc, Cd3d, Cd3e, Il2rg, Prkdc, Sirpa, Foxn1). In anotherembodiment, the rat cell is a somatic cell.

The methods of the present invention can be used to mutate anyeukaryotic cell, including, but not limited to, haploid (in the case ofmultiple gene mutations), diploid, triploid, tetraploid, or aneuploid.In one embodiment, the cell is diploid. Cells in which the methods ofthe present invention can be advantageously used include, but are notlimited to, primary cells (e.g., cells that have been explanted directlyfrom a donor organism) or secondary cells (e.g., primary cells that havebeen grown and that have divided for some period of time in vitro, e.g.,for 10-100 generations). Such primary or secondary cells can be derivedfrom multi-cellular organisms, or single-celled organisms. The cellsused in accordance with the invention include normal cells, terminallydifferentiated cells, or immortalized cells (including cell lines, whichcan be normal, established or transformed), and can be differentiated(e.g., somatic cells or germ cells) or undifferentiated (e.g.,multipotent, pluripotent or totipotent stem cells).

A variety of cells isolated from the above-referenced tissues, orobtained from other sources (e.g., commercial sources or cell banks),can be used in accordance with the invention. Non-limiting examples ofsuch cells include somatic cells, such as, blood cells (erythrocytes andleukocytes), endothelial cells, epithelial cells, neuronal cells (fromthe central or peripheral nervous systems), muscle cells (includingmyocytes and myoblasts from skeletal, smooth or cardiac muscle),connective tissue cells (including fibroblasts, adipocytes,chondrocytes, chondroblasts, osteocytes and osteoblasts) and otherstromal cells (e.g., macrophages, dendritic cells, thymic nurse cells,Schwann cells, etc.). Eukaryotic germ cells (spermatocytes and oocytes)can also be used in accordance with the invention, as can theprogenitors, precursors and stem cells that give rise to theabove-described somatic and germ cells. These cells, tissues and organscan be normal, or they can be pathological such as those involved indiseases or physical disorders, including but not limited to immunerelated diseases (Severe Combined Immunodeficiency (SCID): autosomalrecessive, with sensitivity to ionizing radiation, microchephaly, growthretardation, X-linked, Bare Lymphocyte syndrome) infectious diseases(caused by bacteria, fungi or yeast, viruses (including HIV) orparasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, schizophrenia, musculardystrophy, multiple sclerosis, etc.), or in carcinogenesis and othercancer-related processes. Rat pluripotent cells, including embryoniccells, spermatogonial stem cells, embryonic stem cells, and iPS cellsare envisioned. Rat somatic cells are also envisioned.

In certain embodiments of the invention, cells can be mutated within theorganism or within the native environment as in tissue explants (e.g.,in vivo or in situ). Alternatively, tissues or cells isolated from theorganism using art-known methods and genes can be mutated according tothe present methods. The tissues or cells are either maintained inculture (e.g., in vitro), or re-implanted into a tissue or organism(e.g., ex vivo).

The invention also includes a non-human genetically modified or chimericrat whose genome comprises two chromosomal alleles of a Severe CombinedImmunodeficiency (SCID) gene, wherein at least one of the two allelescontains a mutation, or the progeny of the animal, or an ancestor of theanimal, at an embryonic stage (preferably the one-cell, or fertilizedoocyte stage, and generally, not later than about the 8-cell stage)contains a mutation. The invention also includes the embodiment whereinthe SCID gene of the rat is the Ada gene. In another embodiment, theSCID gene is one of several known SCID genes, such as (Rag1, Rag2,Dclre1c, Nhej1, Jak3, Il7r, Ptprc, Cd3d, Cd3e, Il2rg, Prkdc Sirpa,Foxn1). The invention is also directed to the embodiment wherein theanimal cell is a rat pluripotent cell. The invention is also directed tothe embodiment wherein the animal cell is a rat somatic cell.

In one embodiment, the SCID gene is mutated directly in the germ cellsof a living organism. The separate transgenes for DNA transposonflanking ends and transposase are facilitated to create an active DNAtransposon which integrates into the rat's genome. A plasmid containingtranposon inverted repeats is used to create the transgenic “donor” rat.A plasmid containing transposase is used to create a separate transgenic“driver” rat. The donor rat is then bred with the driver rat to producea rat which contains both donor transposon with flanking repeats anddriver transposase (FIG. 2). This rat known as the “seed” rat has anactivated DNA transposase which drives transposition events. The seedrat is bred to wild type rats to create heterozygote progeny with newtransposon insertions. The heterozygotes can be interbred to createhomozygous rats. Transposon insertion mutations are identified andrecovered via a cloning and sequencing strategy involving thetransposon-cellular DNA junction fragments. The rats that are identifiedto have a new DNA transposon insertion in a known gene or EST or DNAsequence of interest are called knockout rats.

In one embodiment, the SCID gene is mutated in the oocyte before fusionof the pronuclei. This method for genetic modification of rats usesmicroinjected DNA into the male pronucleus before nuclear fusion. Themicroinjected DNA creates a genetically modified founder rat. A femalerat is mated and the fertilized eggs are flushed from their oviducts.After entry of the sperm into the egg, the male and female pronuclei arcseparate entities until nuclear fusion occurs. The male pronucleus islarger are can be identified via dissecting microscope. The egg can beheld in place by micromanipulation using a holding pipette. The malepronucleus is then microinjected with DNA that can be geneticallymodified. The microinjected eggs are then implanted into a surrogatepseudopregnant female which was mated with a vasectomized male foruterus preparation. The foster mother gives birth to geneticallymodified animal. The microinjection method can introduce geneticmodifications directly to the germline of a living animal.

In another embodiment, the SCID gene is mutated in a pluripotent cell.These pluripotent cells can proliferate in cell culture and begenetically modified without affecting their ability to differentiateinto other cell types including germline cells. Genetically modifiedpluripotent cells from a donor can be microinjected into a recipientblastocyst, or in the case of spermatogonial stem cells can be injectedinto the rete testis of a recipient animal. Recipient geneticallymodified blastocysts are implated into pseudopregnant surrogate females.The progeny which have a genetic modification to the germline can thenbe established, and lines homozygous for the genetic modification can beproduced by interbreeding.

In another embodiment, the SCID gene is mutated in a somatic cell andthen used to create a genetically modified animal by somatic cellnuclear transfer. Somatic cell nuclear transfer uses embryonic, fetal,or adult donor cells which are isolated, cultured, and/or modified toestablish a cell line. Individual donor cells are fused to an enucleatedoocyte. The fused cells are cultured to blastocyst stage, and thentransplanted into the uterus of a pseudopregnant female.

In one embodiment, the present invention is directed to methods formutating a single gene or multiple genes (e.g., two or more) ineukaryotic cells and multicellular organisms. The present inventioncontemplates several methods for creating mutations in the SCID gene(s).In one embodiment the mutation is an insertion mutation. In anotherembodiment the mutation is a deletion mutation. In another embodimentthe method of mutation is the introduction of a cassette or gene trap byrecombination. In another embodiment a small nucleic acid sequencechange is created by mutagenesis (through the creation of frame shifts,stop mutations, substitution mutations, small insertion mutations, smalldeletion mutations, and the like). In yet another embodiment, atransgene is delivered to knockout or knockdown the products of the SCIDgene (mRNA or protein) in trans.

The invention also is directed to insertional mutagens for making themutant cells and organisms, and which also can be used to analyze themutations that are made in the cells and organisms. The invention alsois directed to methods in which one or more mutated genes is tagged by atag provided by the insertional mutagen to allow the detection,selection, isolation, and manipulation of a cell with a genome tagged bythe insertional mutagen and allows the identification and isolation ofthe mutated gene(s). The invention provides methods for making multiplemutations (i.e., mutations in two or more genes that produce a phenotypecumulatively) in cells and organisms and tagging at least one of themutated genes such that the mutation can be rapidly identified.

The term gene disruption as used herein refers to a gene knock-out orknock-down in which an insertional mutagen is integrated into anendogenous gene thereby resulting expression of a fusion transcriptbetween endogenous exons and sequences in the insertional mutagen.

In one embodiment, the invention provides for insertional mutagenesisinvolving the integration of one or more polynucleotide sequences intothe genome of a cell or organism to mutate one or more endogenous genesin the cell or organism. Thus, the insertional mutagenic polynucleotidesof the present invention are designed to mutate one or more endogenousgenes when the polynucleotides integrate into the genome of the cell.

Accordingly, the insertional mutagens used in the present invention cancomprise any nucleotide sequence capable of altering gene expressionlevels or activity of a gene product upon insertion into DNA thatcontains the gene. The insertional mutagens can be any polynucleotide,including DNA and RNA, or hybrids of DNA and RNA, and can besingle-stranded or double-stranded, naturally occurring or non-naturallyoccurring (e.g., phosphorothioate, peptide-nucleic acids, etc.). Theinsertional mutagens can be of any geometry, including but not limitedto linear, circular, coiled, supercoiled, branched, hairpin, and thelike, and can be any length capable of facilitating mutation, andtagging of an endogenous gene. In certain embodiments, the insertionalmutagens can comprise one or more nucleotide sequences that provide adesired function.

In another embodiment, the method further involves transforming a cellwith a nucleic acid construct comprising donor DNA. An example of donorDNA may include a DNA transposon. Transposable elements are discretesequences in the genome which are mobile. They have the ability totranslocate from one position in the genome to another. Unlike mostgenetic entities that can create modification to an organism's genome,transposons do not require homology with the recipient genome forinsertion. Transposons contain inverted terminal repeats which arerecognized by the protein transposase. Transposase facilitates thetransposition event. Transposition can occur in replicative (the elementis duplicated) or nonreplicative (element moves from one site to anotherand is conserved) mechanism. Transposons can either contain their owntransposase or transposase can be added in trans to facilitatetransposition. The transposon promotes genetic modifications in manyways. The insertion itself may cause genetic modification by disruptionof a DNA sequence or introduction of DNA. The transposon may be used todeliver a gene trap.

In another embodiment, the method for mutagenesis involves transforminga cell with nucleic acid by use of a LTR retrotransposon with reversetranscriptase. The retrotransposon is initially composed of a singlestrand of RNA. This single stranded RNA is converted into a doublestranded DNA by reverse transcriptase. This is a linear duplex of DNAthat is integrated into the host's genome by the enzyme integrase. Thisinsertion event is much like a transposition event and can be engineeredto genetically modify a host's genome.

In another embodiment, the method for mutageneis is a non-LTRretrotransposon. Long Interspersed Nucleotide Elements (LINES) areretrotransposons that do not have long terminal repeats (LTR's). TheLINES open reading frame 1 (ORF1) is a DNA binding protein, ORF2provides both reverse transcriptase and endonuclease activity. Theendonucleolytic nick provides the 3 ‘-OH end required for priming thesynthesis of cDNA on the RNA template by reverse transcriptase. A secondcleavage site opens the other strand of DNA. The RNA/DNA hybridintegrates into the host genome before or after converting into doublestranded DNA. The integration process is called target primed reversetranscription (TPRT).

In another embodiment a retrovirus may be used for insertional geneticmodification. The retroviral vector (e.g., lentivirus) inserts itselfinto the genome. The vector can carry a transgene or can be used forinsertional mutagenesis. The infected embryos are then injected into areceptive female. The female gives birth to founder animals which havegenetic modifications in their germline. Genetically modified lines areestablished with these founder animals.

In another embodiment, mutagenesis by recombination of a cassette intothe genome may be facilitated by targeting constructs or homologousrecombination vectors. Homologous recombination vectors are composed offragments of DNA which are homologous to target DNA. Recombinationbetween identical sequences in the vector and chromosomal DNA willresult in genetic modification. The vector may also contain a selectionmethod (e.g., antibiotic resistance or GFP) and a unique restrictionenzyme site used for further genetic modification. The targeting vectorwill insert into the genome at a position (e.g, exon, intron, regulatoryelement) and create genetic modification.

In another embodiment, mutagenesis through recombination of a cassetteinto the genome may be carried out by Serine and Tyrosine recombinasewith the addition of an insertion cassette. Site-specific recombinationoccurs by recombinase protein recognition of DNA, cleavage and rejoiningas a phosphodiesterase bond between the serine or tyrosine residues. Acassette of exogenous or endogenous DNA may be recombined into theserine or tyrosine site. The cassette can contain a transgene, genetrap, reporter gene or other exogenous or endogenous DNA.

In one embodiment, the present invention is directed to methods for bothtargeted (site-specific) DNA insertions and targeted DNA deletions. Inone embodiment, the method involves transformation of a cell with anucleic acid or mRNA construct minimally comprising DNA encoding achimeric zinc finger nuclease (ZFN), which can be used to create a DNAdeletion. In another embodiment, a second DNA construct can be providedthat will serve as a template for repair of the cleavage site byhomologous recombination. In this embodiment, a DNA insertion may becreated. The DNA insertion may contain a gene trap cassette.

The invention also is directed to nucleic acid sequence mutation formaking the mutant cells and organisms.

In one embodiment, the method involves chemical mutagenesis withmutagens such as methane-sulfonic acid ethylester (EMS),N-ethyl-N-nitrosourea (ENU), diepoxyoctane and UV/trimethylpsorlalen tocreate nucleic acid sequence mutations.

In another embodiment, sequence editing methods are used that involvethe delivery of small DNA fragments, hybrid DNA/RNA molecules, andmodified DNA polymers to create sequence mismatches and nucleic acidmutations. RNA/DNA hybrids are molecules composed of a central stretchof DNA flanked by short RNA sequences that form hairpin structures. TheRNA/DNA hybrids can produce single base-pair substitutions and deletionsresulting in nucleotide mutations. Some other sequence editing examplesinclude triplex forming oligonucliotides, small fragment homologousreplacement, single-stranded DNA oligonucleotides, and adeno-associatedvirus (AAV) vectors.

The invention also is directed to genetic expression modification ormutagenesis, which may be carried out by delivery of a transgene thatworks in trans.

In one embodiment, RNA interference (RNAi) may be used to alter theexpression of a gene. Single stranded mRNA can be regulated by thepresence of sections of double stranded RNA (dsRNA) or small interferingRNA (siRNA). Both anti-sense and sense RNAs can be effective ininhibiting gene expression. siRNA mediates RNA interference and iscreated by cleavage of long dsDNA by the enzyme Diccr. RNAi can creategenetic modification by triggering the degradation of mRNA's that arecomplementary to either strand of short dsRNA. When siRNA is associatedwith complementary single-stranded RNA it can signal for nuclease todegrade the mRNA. RNAi can also result in RNA silencing which occurswhen the short dsRNA inhibits expression of a gene. Other forms ofinhibitory RNA, such as small hairpin RNA (shRNA) are envisioned.

In another embodiment, the delivery of a transgene encoding a dominantnegative protein may alter the expression of a target gene. Dominantnegative proteins can inhibit the activity of an endogenous protein. Oneexample is the expression a protein which contains the ligand bindingsite of an endogenous protein. The expressed dominant-negative protein“soaks up” all of the available ligand. The endogenous protein istherefore not activated, and the wild type function is knocked out orknocked down.

Other schemes based on these general concepts are within the scope andspirit of the invention, and are readily apparent to those skilled inthe art.

The invention also provides methods for making homozygous mutations inrats by breeding a genetically modified rat which is heterozygous for amutant allele with another genetically modified rat which isheterozygous for the same mutant allele. On average 25% of offspring ofsuch matings are expected to produce animals that are homozygous for themutant allele. Homozygous mutations are useful for discovering functionsassociated with the mutated gene.

The present invention is directed generally to reduction or inactivationof gene function or gene expression in cells in vitro and inmulticellular organisms. The invention encompasses methods for mutatingcells using one or more mutagens, particularly wherein at least onemutation is an insertion mutation, a deletion mutation, or a nucleicacid sequence mutation, to achieve a homozygous gene mutation ormutation of multiple genes required cumulatively to achieve a phenotype.The methods are used to create knock-outs, knock-downs, and othermodifications in the same cell or organism.

The mutation can result in a change in the expression level of a gene orlevel of activity of a gene product. Activity encompasses all functionsof a gene product, e.g., structural, enzymatic, catalytic, allosteric,and signaling. In one embodiment, mutation results in a decrease orelimination of gene expression levels (RNA and/or protein) or a decreaseor elimination of gene product activity (RNA and/or protein). Mostmutations will decrease the activity of mutated genes. However, both theinsertional and physicochemical mutagens can also act to increase or toqualitatively change (e.g., altered substrate on binding specificity, orregulation of protein activity) the activity of the product of themutated gene. Although mutations will often generate phenotypes that maybe difficult to detect, most phenotypically detectable mutations changethe level or activity of mutated genes in ways that are deleterious tothe cell or organism.

As used herein, decrease means that a given gene has been mutated suchthat the level of gene expression or level of activity of a gene productin a cell or organism is reduced from that observed in the wild-type ornon-mutated cell or organism. This is often accomplished by reducing theamount of mRNA produced from transcription of a gene, or by mutating themRNA or protein produced from the gene such that the expression productis less abundant or less active.

Disclosed are cells produced by the process of transforming the cellwith any of the disclosed nucleic acids. Disclosed are cells produced bythe process of transforming the cell with any of the non-naturallyoccurring disclosed nucleic acids.

Disclosed are any of the disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thenon-naturally occurring disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thedisclosed peptides produced by the process of expressing any of thenon-naturally disclosed nucleic acids.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules disclosedherein. Disclosed are animals produced by the process of transfecting acell within the animal any of the nucleic acid molecules disclosedherein, wherein the animal is a rat. Also disclosed are animals producedby the process of transfecting a cell within the animal any of thenucleic acid molecules disclosed herein, wherein the mammal is a rat.

Such methods are used to achieve mutation of a single gene to achieve adesired phenotype as well as mutation of multiple genes, requiredcumulatively to achieve a desired phenotype, in a rat cell or rat. Theinvention is also directed to methods of identifying one or more mutatedgenes, made by the methods of the invention, in rat cells and in rats,by means of a tagging property provided by the insertional mutagen(s).The insertional mutagen thus allows identification of one or more genesthat are mutated by insertion of the insertional mutagen.

The invention is also directed to rat cells and rats created by themethods of the invention and uses of the rat cells and rats. Theinvention is also directed to libraries of rat cells created by themethods of the invention and uses of the libraries.

Severe Combined Immunodeficiency (SCID)-Associated Genes

The invention also features a novel genetically modified rat with agenetically engineered modification in a gene encoding a Severe CombinedImmunodeficiency (SCID)-associated protein. In another aspect, theinvention features a genetically modified rat, wherein a gene encodingSCID protein is modified resulting in reduced SCID protein activity. Inpreferred embodiments of this aspect, the genetically modified rat ishomozygous for the modified gene. In other preferred embodiments, thegene encoding SCID protein is modified by disruption, and thegenetically modified rat has reduced SCID protein activity. In yetanother embodiment, the transgenic rat is heterozygous for the genemodification.

In another embodiment of this aspect of the invention, the inventionfeatures a nucleic acid vector comprising nucleic acid capable ofundergoing homologous recombination with an endogenous SCID gene in acell, wherein the homologous recombination results in a modification ofthe SCID gene resulting in decreased SCID protein activity in the cell.In another aspect, the modification of the SCID gene is a disruption inthe coding sequence of the endogenous SCID gene.

Another embodiment of this aspect of the invention features a rat cell,wherein the endogenous gene encoding SCID protein is modified, resultingin reduced SCID protein activity in the cell.

In certain embodiments, the reduced SCID protein activity is manifested.In a related aspect, the invention features a rat cell containing anendogenous SCID gene into which there is integrated a transposoncomprising DNA encoding a gene trap and/or a selectable marker.

In another aspect, the invention features a rat cell containing anendogenous SCID gene into which there is integrated a retrotransposoncomprising DNA encoding a gene trap and/or a selectable marker. Inanother aspect, the invention features a rat cell containing anendogenous SCID gene into which there is DNA comprising an insertionmutation in the SCID gene. In another aspect, the invention features arat cell containing an endogenous SCID gene into which there is DNAcomprising a deletion mutation in the SCID gene. In another aspect, theinvention features a rat cell containing an endogenous SCID gene inwhich there has been nucleic acid sequence modification of the SCIDgene.

In another embodiment of the invention, the invention features a methodfor determining whether a compound is potentially useful for treating oralleviating the symptoms of a SCID gene disorder, which includes (a)providing a cell that produces a SCID protein, (b) contacting the cellwith the compound, and (c) monitoring the activity of the SCID protein,such that a change in activity in response to the compound indicatesthat the compound is potentially useful for treating or alleviating thesymptoms of a SCID gene disorder.

It is understood that simultaneous targeting of more than one gene maybe utilized for the development of “knock-out rats” (i.e., rats lackingthe expression of a targeted gene product), “knock-in rats” (i.e., ratsexpressing a fusion protein or a protein encoded by a gene exogenous tothe targeted locus), “knock down rats” (i.e., rats with a reducedexpression of a targeted gene product), or rats with a targeted genesuch that a truncated gene product is expressed.

Rat models that have been genetically modified to alter SCID geneexpression may be used in in vivo assays to test for activity of acandidate SCID modulating agent, or to further assess the role of SCIDgene in a (SCID pathway process such as V(D)J recombination or NaturalKiller (NK) cell activity. Preferably, the altered SCID gene expressionresults in a detectable phenotype, such as decreased levels of T-, B-,and Natural Killer (NK)-cells, non-homologous end joining (NHEJ)function, or and increase in susceptibility to infections compared tocontrol animals having normal SCID gene expression. The geneticallymodified rat may additionally have altered SCID gene expression (e.g.,SCID gene knockout). In one embodiment, the genetically modified ratsare genetically modified animals having a heterologous nucleic acidsequence present as an extrachromosomal element in a portion of itscells, i.e. mosaic animals (see, for example, techniques described byJakobovits, 1994, Curr. Biol. 4:761-763) or stably integrated into itsgerm line DNA (i.e., in the genomic sequence of most or all of itscells). Heterologous nucleic acid is introduced into the germ line ofsuch genetically modified animals by genetic manipulation of, forexample, embryos or germ cells or germ cells precursors of the hostanimal.

Methods of making genetically modified rodents are well-known in the art(see Brinster ct al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985),U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat.No. 4,873,191 by Wagner et al., and Hogan, B., Manipulating the MouseEmbryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1986); for particle bombardment see U.S. Pat. No. 4,945,050, bySandford et al.; for genetically modified Drosophila see Rubin andSpradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; forgenetically modified insects see Berghammer A. J. et al., A UniversalMarker for Genetically modified Insects (1999) Nature 402:370-371; forgenetically modified Zebrafish see Lin S., Genetically modifiedZebrafish, Methods Mol Biol. (2000); 136:375-3830); for microinjectionprocedures for fish, amphibian eggs and birds see Houdebine andChourrout, Experientia (1991) 47:897-905; Hammer et al., Cell (1990)63:1099-1112; and for culturing of embryonic stem (ES) cells and thesubsequent production of genetically modified animals by theintroduction of DNA into ES cells using methods such as electroporation,calcium phosphate/DNA precipitation and direct injection see, e.g.,Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J.Robertson, ed., IRL Press (1987)). Clones of the nonhuman geneticallymodified animals can be produced according to available methods (seeWilmut, I. et al. (1997) Nature 385:810-813; and PCT InternationalPublication Nos. WO 97/07668 and WO 97/07669).

In one embodiment, the genetically modified rat is a “knock-out” animalhaving a heterozygous or homozygous alteration in the sequence of anendogenous SCID gene that results in a decrease of immune function,preferably such that SCID gene expression is undetectable orinsignificant. Knock-out animals are typically generated by homologousrecombination with a vector comprising a transgene having at least aportion of the gene to be knocked out. Typically a deletion, addition orsubstitution has been introduced into the transgene to functionallydisrupt it. The transgene can be a human gene (e.g., from a humangenomic clone) but more preferably is an ortholog of the human genederived from the genetically modified host species. For example, a mouseSCID gene is used to construct a homologous recombination vectorsuitable for altering an endogenous SCID gene in the mouse genome.Detailed methodologies for homologous recombination in rodents areavailable (see Capecchi, Science (1989) 244:1288-1292; Joyner et al.,Nature (1989) 338:153-156). Procedures for the production of non-rodentgenetically modified mammals and other animals are also available(Houdebine and Chourrout, supra; Pursel et al., Science (1989)244:1281-1288; Simms et al., Bio/Technology (1988) 6:179-183). In apreferred embodiment, knock-out animals, such as rats harboring aknockout of a specific gene, may be used to produce antibodies againstthe human counterpart of the gene that has been knocked out (Claesson MHet al., (1994) Scan J Immunol 40:257-264; Declerck P J et al., (1995) JBiol Chem. 270:8397-400).

In another embodiment, the genetically modified rat is a “knock-down”animal having an alteration in its genome that results in alteredexpression (e.g., decreased expression) of the SCID gene, e.g., byintroduction of mutations to the SCID gene, or by operatively insertinga regulatory sequence that provides for altered expression of anendogenous copy of the SCID gene.

Genetically modified rats can also be produced that contain selectedsystems allowing for regulated expression of the transgene. One exampleof such a system that may be produced is the cre/loxP recombinase systemof bacteriophage P1 (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat.No. 4,959,317). If a cre/loxP recombinase system is used to regulateexpression of the transgene, animals containing transgenes encoding boththe Cre recombinase and a selected protein are required. Such animalscan be provided through the construction of “double” geneticallymodified animals, e.g., by mating two genetically modified animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase. Another example of arecombinase system is the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No.5,654,182). In a preferred embodiment, both Cre-LoxP and Flp-Frt areused in the same system to regulate expression of the transgene, and forsequential deletion of vector sequences in the same cell (Sun X et al(2000) Nat Genet 25:83-6).

The genetically modified rats can be used in genetic studies to furtherelucidate the immune function pathways, as animal models of disease anddisorders implicating defective immune function, and for in vivo testingof candidate therapeutic agents, such as those identified in screensdescribed below. The candidate therapeutic agents are administered to agenetically modified animal having altered immune system and phenotypicchanges are compared with appropriate control animals such asgenetically modified animals that receive placebo treatment, and/oranimals with unaltered immune systems that receive candidate therapeuticagent.

The invention also features novel genetically modified animals with agenetically engineered modification in the gene encoding SCID proteins.In one aspect, the invention features a genetically modified non-humanmammal, wherein a gene encoding SCID gene is provided as follows:

T-cell(−), B-cell (−), natural killer (NK)-cell (−) SCID: Ada

The Ada gene encodes the enzyme Adenosine deaminase which catalyzes thehydrolysis of adenosine and deoxyadenosine to inosine in the purinecatabolic pathway. Defects in Ada lead to SCID in which the B-, T-, andNK-cells are depleted. In humans, ADA deficiency accounts for 15% of allSCID, 30% autosomal recessive SCID, and 50% of non-X-linked SCID. Thereare multiple forms of SCID; early-,delayed-, and late-onset. Early-onsetbeing the most common and with symptoms occurring immediately afterbirth. Delayed-onset constitutes 10-15% of SCID due to ADA deficiencyand symptoms occur between 6-24 months after birth. Late-onset symptomsoccur between 4 years of age and into adulthood. Partial SCID occurswhen an individual has decreased ADA activity in erythrocytes, butposses up to 80% of the normal number of leukocytes. Common SCIDsymptoms include recurrent respiratory and other organ infectionsresulting in severe inflammation and insufficiency leading ultimately todeath.

By the methods of cell hybridization, Southern blot, gene dosage, andhigh-resolution in situ hybridization, the rat Ada locus was mapped toposition 3q42. In Ada deficient cells, immature thymocytes undergoapoptosis. Adenosine deaminase catalyzes the hydrolysis of adenosine anddeoxyadenosine triphosphate (dATP) to inosine. The accumulation of dATPand adenosine inhibits ribonucleotide reductase's ability to reducepurine and pyrimidine ribonucleotides, which is a step required for DNAsynthesis. The accumulation of dATP and adenosine also inhibitsS-adenosylhomocysteine hydrolase, which is required for cell viability.Both consequences of Ada deficiency are lymphotoxic and result indecreased or absent T-, B-, and NK cells. However, it has been shownthat inhibition of adenosine kinase provides T-cell recovery, and that aphosphorylated ADA substrate is the cause of lymphotoxicity. It has alsobeen suggested that dATP accumulation induces cytochrome c release fromthe mitochondria and initiates apoptosis of thymocytes.

T-cell (−), B-cell (−), NK-cell (+) SCID: Rag1 & Rag2

Recombination activating genes 1 & 2 (Rag1 &Rag2) play an essential rolein the activation of immunoglobin V(D)J recombination to create variableimmunoglobins and T-cell receptors. The proteins encoded by the geneswork by creating nicks in DNA at conserved recombination signalsequences (RSS). The nicks create double-stranded breaks that then forma covalently sealed hairpin intermediate and recombination throughsignal joint formation commences. The recombination activating genes areessential for immunoglobin and T-cell receptor assembly from developinglymphocytes. It has been shown that RAG1 specific amino acid changeslead to competent DNA cleavage, but result in defective in signal jointformation. Rag1 interacts directly with DNA. RAG2 contains geneticelements on its 5-prime end which coordinate expression of Rag1 & Rag2.RAG2 plays an essential role in recognition and cleavage of distortedDNA intermediates and is critical in the joining step in V(D)Jrecombination. RAG2 also interacts with histone H3K4me3 which plays animportant role in V(D)J recombination. Mutations to the active site ofRAG2 and H3K4me3 impair V(D)J recombination. The recombinationactivating proteins have a special non-homologous end joining (NHEJ)function. In NHEJ−/− deficient cells, RAG1 & RAG2 interfaces with NHEJfactors to maintain NHEJ function and integrity.

In humans, 20-30% of all SCID cases are T-cell (−), B-cell (−), NK-cell(+), and within this group 50% have mutations to RAG1 or RAG2. Typicallypatients are admitted to the hospital before 100 days of life. SCIDpatients undergo recurrent diarrhea, fever, candidiasis, lung infectionsand other infections. If not treated by stem cell therapy or genetherapy the SCID is always fatal. Disruption of RAG1 & RAG2 V(D)Jrecombination function leads to the arrest of T- and B-cell developmentand immune system failure.

Omenn Syndrome

Omenn Syndrome is a less severe immune deficiency than SCID,characterized by autoimmune responses resulting in hepatomegaly, andsplenomegaly. Some other features are reticuloendotheliosis, skindisorders, diarrhea, and often terminal leukemia. In several Omennsyndrome cases the RAG2 recognition site for Histone H3K4me3 is mutated.In Omenn patients, the autoimmune regulator AIRE is downregulated, andno insulin self-antigen, or cytochrome p450 1A2 exist.

T-cell (−), B-cell (−), NK-cell (−) with sensitivity to ionizingradiation SCID: Dclre1c & Prkdc

RT-PCR and BAC contig analysis mapped the Dclre1c gene which encodes theprotein Artemis to locus 17q12.3 in rat. Dclre1c activity is criticalfor V(D)J recombination and DNA repair. Artemis has 5′-to3′ exonucleaseactivity alone, but also acquires 5′-to3′ endonuclease activity when incomplex and phosphorylated by Prkdc. The complex formed between thesetwo proteins is critical for the hairpin opening step of V(D)Jrecombination, and plays a role in NHEJ.

Mutations to the Dclre1c gene results in Athabaskan-type SCID (SCIDA)and partial SCID. SCIDA exhibits sensitivity to ionizing radiation. Thissensitivity is due to the lack of DNA repair machinery activity. Thesymptoms are similar to other SCID phenotypes with continuousinfections, diarrhea, fever, lymphopenia. However, in partial SCID lowlevels of polyclonal T- and B-cells are found. Partial SCID was found tobe the result of a hypomorphic mutation to Artemis.

The Prkdc gene is a nuclear DNA-dependent serine/threonine proteinkinase which must interact with autoimmune antigen Ku and be bound toDNA in order to exhibit it catalytic properties. The Prkdc gene wasmapped by in situ hybridization to locus 11q23 in rats. Prkdc binds toDNA double strand breaks and other cleavages that occur after damage orduring recombination intermediates. Prkdc−/− cells are hypersensitive todamage by ionizing radiation due to their inability to repair doublestrand breaks. In Prkdc−/− cells, V(D)J intermediates are unable to beprocessed and ligated. Therefore, B- and T-cells do not develop matureimmunoglobulin and T-cell receptors.

T-cell (−), B-cell (−), NK-cell (+) with microcephaly, growthretardation, and sensitivity to ionizing radiation: Nhej1

The Nhej1 gene encodes a DNA repair factor Xlf which is structurallysimilar to Xrcc4. Nhej1 was mapped to the 9q33 locus in rats. Xlfinteracts with Xrcc4 and Lig4 and is essential for the non-homologousend joining (NHEJ) pathway. Nhej1 −/− defective cells and rats exhibitionizing radiation sensitivity and the inability to undergo V(D)Jrecombination. Mutations to the Nhej1 gene result in B-, T-cell, andCd45ro memory T-cell deficiency, but natural killer (NK) cell levels andactivity remain normal. Disruption in the Nhej1 gene in humans leads torecurrent infections, hypogammaglobulinemia, and B- and T-celllymphocytopenia.

T-cell (−), B-cell (+), NK-cell (−) SCID: Jak3& Il2rg

The Jak3 gene produces a tyrosine kinase that is involved in cytokinereceptor-mediated intracellular signal transduction. In situhybridization studies have been used to mapped the gene to locus 16p14in rats. Interleukin2 gamma-c receptor (I12gr) induces phosphorylationand activation of Jak3. Il2gr and Jak3 operate through the same pathwayand share this pathway with multiple cytokines. I12 is a T-cell growthfactor which is critical for the development of active T-celllymphocytes. Mutations that decrease the association between I12rg andJak3 result in X-linked SCID. Inhibition of Jak3 or I12rg disrupts thesignal pathway and results in early and severe T-cell and NK-celldevelopmental blocking.

Humans that have a disruption in the JAK3 or IL3RG genes or theJAK3/IL2GR pathway have T-cell (−), B-cell (+), NK-cell (−) SCID.Patients have an absence of mature circulating T-lymphocytes andNK-cells, a normal level of nonfunctional B-cells, and hypoplasia inlymphoid tissues.

T-cell (−), B-cell (+), NK-cell (+), with decreased immunoglobulins:Il7r, Cd45, Cd3d, Cd3e

The Il7r gene encodes Interleukin receptor7 which requires Il2rg forproper function. The Il7r gene was mapped to the 2q16 locus in ratsusing in situ hybridization and Southern blot analysis. Il7 interactswith Tslp to enhance dendritic cell (DC) monocyte maturation whichinduces T-cell proliferation. Memory T-cell lymphocytes that expresshigh levels of Il7r also express high levels of anti-apoptotic markers.A disruption in the Il7r gene leads to increased apoptosis of T-cells.Il7−/− cells exhibit decreased V(H)-D(H)-J(H) joining.

The Ptprc gene or Cd45 encodes a protein that is involved in cellgrowth, differentiation, and mitotic cycle regulation. The gene wasmapped to the 13q13 locus in rats. The Cd45 protein suppresses Jakkinases, and plays a role in cytokine signaling. The Cd45 protein isessential for activation of T-cells mediated by the cell-to-cell,receptor-antigen signal transduction pathway. Cd45−/− undergo a block ofT-cell maturation.

The Cd3d gene was mapped to the locus 8q22 in rats. The Cd3d protein isessential for T-cell development and signal transduction. In Cd3d−/−cells immature thymocytes are negatively selected and removed beforedifferentiation into mature thymocytes. T-cell differentiation isblocked at early stage entry.

Through somatic cell hybrids and in situ hybridization, the Cd3e genewas mapped to locus 8q22 in rats. The Cd3e gene encodes a protein thatforms a T-cell receptor complex. The Cd3e protein is essential forT-cell antigen recognition and intracellular signaling transductionpathways.

Humans with deficient IL7R, CD45, CD3D, CD3E genes have T-cell (−),B-cell (+), NK-cell (+) SCID. Fever, rash, hepatosplenomegaly,lymphadenopathy, pneumonitis, pancytopenia, and cytomegalovirusinfection are common symptoms among patients. Patients display a lowT-cell count, decreased immunoglobulins, and abnormal CD45, CD3D, CD3Eexpression.

The invention also features novel genetically modified cells and animalswith a genetically engineered modification in a gene encoding SCIDproteins. In one aspect, the invention features genetically modified ratcells or rats, wherein a gene modification occurs in a gene encoding aSCID protein provided in Table 1:

TABLE 1 Rat Chromosomal SCID gene Function Location Ada Hydrolysis ofadenosine 3q42 NM_130399.2 and deoxyadenosine triphosphate (dATP) toinosine Rag1 Immunoglobin V(D)J 3q31 NM_053468.1 recombination Rag2Immunoglobin V(D)J 3q31 NM_001100528.1 recombination Dclre1c V(D)Jrecombination and 17q12.3 NM_147145.1 DNA repair Prkdc binds to DNAdouble 11q23 NM_001108327.1 strand breaks and other cleavages that occurafter damage or during recombination intermediates, and facilitatesrepair Nhej1 non-homologous end 9q33 NM_001014217.1 joining (NHEJ)pathway Jak3 cytokine receptor mediated 16p14 NM_012855.1 intracellularsignal transduction Il2rg Il2 is a T-cell growth factor Xq31 NM_080889.1which is critical for the development of active T- cell lymphocytes, andinduces phosphorylation and activation of Jak3 Il7r enhances DC monocyte2q16 NM_001106418.1 maturation which induces T-cell proliferation Cd45cell growth, differentiation, 13q13 NM_138507.1 and mitotic cycleregulation Cd3d T-cell development and 8q22 NM_013169.1 signaltransduction Coro1a Chemokine mediated 1q36 NM_130411.2 migration, andlymphocyte development Cd3e T-cell antigen recognition 8q22NM_0001108140.1 and intracellular signaling transduction SirpaSignal-regulatory protein 3q36 NM_013016.2 alpha plays a role inmacrophage fusion Foxn1 Mutations are associated 10q25 NM_00110648.1with congenital athymia and hairlessness

Methods

The methods used in the present invention are comprised of a combinationof genetic introduction methods, genetic modification or mutagenesismechanisms, and vector delivery methods. For all genetic modification ormutagenesis mechanisms one or more introduction and delivery method maybe employed. The invention may include but is not limited to the methodsdescribed below.

Genetic Introduction Methods

In one introduction method, the SCID gene is mutated directly in thegerm cells of an adult animal. This method usually involves the creationof a transgenic founder animal by pronuclear injection. Rat oocytes aremicroinjected with DNA into the male pronucleus before nuclear fusion.The microinjected DNA creates a transgenic founder rat. In this method,a female rat is mated and the fertilized eggs are flushed from theiroviducts. After entry of the sperm into the egg, the male and femalepronuclei are separate entities until nuclear fusion occurs. The malepronucleus is larger are can be identified via dissecting microscope.The egg can be held in place by micromanipulation using a holdingpipette. The male pronucleus is then microinjected with DNA that can begenetically modified. The microinjected eggs are then implanted into asurrogate pseudopregnant female which was mated with a vasectomized malefor uterus preparation. The foster mother gives birth to transgenicfounder animals. If the transgenic DNA encodes the appropriatecomponents of a mutagenesis system, such as transposase and a DNAtransposon, then mutagenesis will occur directly in the germ cells offounder animals and some offspring will contain new mutations. Chemicalmutagenesis can also be used to cause direct germ line mutations.

In another introduction method, the SCID gene is mutated in the earlyembryo of a developing animal. The mutant embryonic cells develop toconstitute the germ cells of the organism, thereby creating a stable andheritable mutation. Several forms of mutageneis mechanisms can beintroduced this way including, but not limited to, zinc finger nucleasesand delivery of gene traps by a retrovirus.

In another introduction method, the SCID gene is mutated in apluripotent cell. These pluripotent cells can proliferate in cellculture and be genetically modified without affecting their ability todifferentiate into other cell types including germ line cells.Genetically modified pluripotent cells from a donor can be microinjectedinto a recipient blastocyst, or in the case of spermatogonial stem cellscan be injected into the rete testis of a recipient animal. Recipientgenetically modified blastocysts are implanted into pseudopregnantsurrogate females. The progeny which have a genetic modification to thegerm line can then be established, and lines homozygous for the geneticmodification can be produced by interbreeding.

In another introduction method, the SCID gene is mutated in a somaticcell and then used to create a genetically modified animal by somaticcell nuclear transfer. Somatic cell nuclear transfer uses embryonic,fetal, or adult donor cells which are isolated, cultured, and/ormodified to establish a cell line. Individual donor cells are fused toan enucleated oocyte. The fused cells are cultured to blastocyst stage,and then transplanted into the uterus of a pseudopregnant female.Alternatively the nucleus of the donor cell can be injected directlyinto the enucleated oocyte. See U.S. Appl. Publ. No. 20070209083.

Genetic Modification Methods

Mobile DNA Technology

DNA transposons are discrete mobile DNA segments that are commonconstituents of plasmid, virus, and bacterial chromosomes. Theseelements arc detected by their ability to transpose self-encodedphenotypic traits from one replicon to another, or to transpose into aknown gene and inactivate it. Transposons, or transposable elements,include a piece of nucleic acid bounded by repeat sequences. Activetransposons encode enzymes (transposases) that facilitate the insertionof the nucleic acid into DNA sequences.

The lifecycle and insertional mutagenesis of DNA transposon SleepingBeauty (SB) is depicted in FIG. 1. In its lifecycle, the SB encodes atransposase protein. That transposase recognizes the inverted terminalrepeats (ITRs) that flank the SB transposon. The transposase thenexcises SB and reintegrates it into another region of the genome.Mutagenesis via Sleeping Beauty is depicted. The mechanism is similar tothe life cycle, but transposase is not encoded by the transposon, butinstead is encoded elsewhere in the genome

The Sleeping Beauty (SB) mutagenesis breeding and screening scheme isdepicted in FIG. 2. One rat referred to as the “driver” rat contains the(SB) transposase within its genome. A second rat, the “donor” ratcontains the transposon which has the transposase-recognizable invertedterminal repeats (ITRs). The two rats are bred to create the “seed” ratwhich has an active transposon containing transposase and ITRs. Thetransposon recognizes the ITRs, excises the transposon, and inserts itelsewhere in the rat's genome. This insertion event often disruptscoding, regulatory, and other functional regions in the genome to createknockout rat models. The “seed” rat is bred with wild type rats whichbeget heterozygous G1 mutants. If the transposon has inserted into thegenome, the event will be recorded via size comparison of DNA bySouthern blot analysis. The exact location of the transposon insertionis determined by PCR-based amplification methods combined withsequencing of the DNA flanking the new insertion.

The sequences for the DNA transposons Sleeping Beauty (SB) piggyBac (PB)functional domains are shown in FIG. 3. The SB and PB transposasesequences encode the protein that recognizes the ITRs and carries outthe excision and re-integration. The 3′ and 5′ ITRs are the flankingsequences which the respective transposases recognizes in order to carryout excision and reintegration elsewhere in the genome.

The DNA transposon Sleeping Beauty (SB) was used by the inventors tocreate a knockout rat in the Ada gene. The mechanism is depicted in FIG.4, and is the same as that described above. The transposase is encoded,and the protein recognizes the ITRs of the transposon. The transposon isthen excised and reinserted into the seventh intron of the rat Ada genewhich resides on chromosome 3, location 3q42.

In another embodiment, the present invention utilizes the transposonpiggyBac, and sequence configurations outside of piggyBac, for use as amobile genetic element as described in U.S. Pat. No. 6,962,810. TheLepidopteran transposon piggyBac is capable of moving within the genomesof a wide variety of species, and is gaining prominence as a useful genetransduction vector. The transposon structure includes a complex repeatconfiguration consisting of an internal repeat (IR), a spacer, and aterminal repeat (TR) at both ends, and a single open reading frameencoding a transposase.

The Lepidopteran transposable element piggyBac transposes via a uniquecut-and-paste mechanism, inserting exclusively at 5′ TTAA 3′ targetsites that are duplicated upon insertion, and excising precisely,leaving no footprint (Elick et al., 1996b; Fraser et al., 1996; Wang andFraser 1993).

In another embodiment, the present invention utilizes the SleepingBeauty transposon system for genome manipulation as described, forexample, in U.S. Pat. No. 7,148,203. In one embodiment, the systemutilizes synthetic, salmonid-type Tc1-like transposases with recognitionsites that facilitate transposition. The transposase binds to twobinding-sites within the inverted repeats of salmonid elements, andappears to be substrate-specific, which could prevent cross-mobilizationbetween closely related subfamilies of fish elements.

In another aspect of this invention, the invention relates to atransposon gene transfer system to introduce DNA into the DNA of a cellcomprising: a nucleic acid fragment comprising a nucleic acid sequencepositioned between at least two inverted repeats wherein the invertedrepeats can bind to a SB protein and wherein the nucleic acid fragmentis capable of integrating into DNA of a cell; and a transposase ornucleic acid encoding a transposase. In one embodiment, the transposaseis provided to the cell as a protein and in another the transposase isprovided to the cell as nucleic acid. In one embodiment the nucleic acidis RNA and in another the nucleic acid is DNA. In yet anotherembodiment, the nucleic acid encoding the transposase is integrated intothe genome of the cell. The nucleic acid fragment can be part of aplasmid or a recombinant viral vector. Preferably, the nucleic acidsequence comprises at least a portion of an open reading frame and alsopreferably, the nucleic acid sequence comprises at least a regulatoryregion of a gene. In one embodiment the regulatory region is atranscriptional regulatory region and the regulatory region is selectedfrom the group consisting of a promoter, an enhancer, a silencer, alocus-control region, and a border element. In another embodiment, thenucleic acid sequence comprises a promoter operably linked to at least aportion of an open reading frame.

In the transgene flanked by the terminal repeats, the terminal repeatscan be derived from one or more known transposons. Examples oftransposons include, but are not limited to the following: SleepingBeauty (Izsvak Z, Ivics Z. and Plasterk R H. (2000) Sleeping Beauty, awide host-range transposon vector for genetic transformation invertebrates. J. Mol. Biol. 302:93-102), mos1 (Bessereau J L, et al.(2001) Mobilization of a Drosophila transposon in the Caenorhabditiselegans germ line. Nature. 413(6851):70-4; Zhang L, et al. (2001)DNA-binding activity and subunit interaction of the mariner transposase.Nucleic Acids Res.29(17):3566-75, piggyBac (Tamura T. et al. Germ linetransformation of the silkworm Bombyx mori L. using a piggyBactransposon-derived vector. Nat Biotcchnol. 2000 Jan; 18(1):81-4), Himar1(Lampe D J, et al. (1998) Factors affecting transposition of the Himar1mariner transposon in vitro. Genetics. 149(11):179-87), Hermes, Tol2element, Pokey, Tn5 (Bhasin A, et al. (2000) Characterization of a Tn5pre-cleavage synaptic complex. J Mol Biol 302:49-63), Tn7 (Kuduvalli PN, Rao J E, Craig N L. (2001) Target DNA structure plays a critical rolein Tn7 transposition. EMBO J 20:924-932), Tn916 (Marra D, Scott J R.(1999) Regulation of excision of the conjugative transposon Tn916. MolMicrobiol 2:609-621), Tc1/mariner (Izsvak Z, Ivics Z4 Hackett P B.(1995) Characterization of a Tc1-like transposable element in zebrafish(Danio rerio). Mol. Gen. Genet. 247:312-322), Minos and S elements(Franz G and Savakis C. (1991) Minos, a new transposable element fromDrosophila hydei, is a member of the Tc1-like family of transposons.Nucl. Acids Res. 19:6646; Merriman P J, Grimes C D, Ambroziak J, HackettD A, Skinner P, and Simmons M J. (1995) S elements: a family of Tc1-liketransposons in the genome of Drosophila melanogaster. Genetics141:1425-1438), Quetzal elements (Ke Z, Grossman G L, Cornel A J,Collins F H. (1996) Quetzal: a transposon of the Tc1 family in themosquito Anopheles albimanus. Genetica 98:141-147); Txr elements (Lam WL, Seo P, Robison K, Virk S, and Gilbert W. (1996) Discovery ofamphibian Tc1-like transposon families. J Mol Biol 257:359-366),Tc1-like transposon subfamilies (Ivics Z, Izsvak Z, Minter A, Hackett PB. (1996) Identification of functional domains and evolution of Tc1-liketransposable elements. Proc. Natl. Acad Sci USA 93: 5008-5013), Tc3 (TuZ. Shao H. (2002) Intra- and inter-specific diversity of Tc-3 liketransposons in nematodes and insects and implications for theirevolution and transposition. Gene 282:133-142), ICESt1 (Burrus V et al.(2002) The ICESt1 element of Streptococcus thermophilus belongs to alarge family of integrative and conjugative elements that exchangemodules and change their specificity of integration. Plasmid. 48(2):77-97), maT, and P-element (Rubin G M and Spradling A C. (1983) Vectorsfor P element-mediated gene transfer in Drosophila. Nucleic Acids Res.11:6341-6351). These references are incorporated herein by reference intheir entirety for their teaching of the sequences and uses oftransposons and transposon ITRs.

Translocation of Sleeping Beauty (SB) transposon requires specificbinding of SB transposase to inverted terminal repeats (ITRs) of about230 bp at each end of the transposon, which is followed by acut-and-paste transfer of the transposon into a target DNA sequence. TheITRs contain two imperfect direct repeats (DRs) of about 32 bp. Theouter DRs are at the extreme ends of the transposon whereas the innerDRs are located inside the transposon, 165-166 bp from the outer DRs.Cui et al. (J. Mol Biol 318:1221-1235) investigated the roles of the DRelements in transposition. Within the 1286-bp element, the essentialregions arc contained in the intervals bounded by coordinates 229-586,735-765, and 939-1066, numbering in base pairs from the extreme 5′ endof the element. These regions may contain sequences that are necessaryfor transposase binding or that are needed to maintain proper spacingbetween binding sites.

Transposons are bracketed by terminal inverted repeats that containbinding sites for the transposase. Elements of the IR/R subgroup of theTc1/mariner superfamily have a pair of transposase-binding sites at theends of the 200-250 bp long inverted repeats (IRs) (Izsvak, et al.1995). The binding sites contain short, 15-20 bp direct repeats (DRs).This characteristic structure can be found in several elements fromevolutionarily distant species, such as Minos and S elements in flies(Franz and Savakis, 1991; Merriman et al, 1995), Quetzal elements inmosquitoes (Ke et al, 1996), Txr elements in frogs (Lam et al, 1996) andat least three Tc1 like transposon subfamilies in fish (Ivics et al.,1996), including SB [Sleeping Beauty] and are herein incorporated byreference.

Whereas Tc1 transposons require one binding site for their transposasein each IR, Sleeping Beauty requires two direct repeat (DR) bindingsites within each IR, and is therefore classified with Tc3 in an IR/DRsubgroup of the Tc1 /mariner superfamily (96,97). Sleeping Beautytransposes into TA dinucleotide sites and leaves the Tc1/marinercharacteristic footprint, i.e., duplication of the TA, upon excision.The non-viral plasmid vector contains the transgene that is flanked byIR/DR sequences, which act as the binding sites for the transposase. Thecatalytically active transposase may be expressed from a separate(trans) or same (cis) plasmid system. The transposase binds to theIR/DRs, catalyzes the excision of the flanked transgene, and mediatesits integration into the target host genome.

Naturally occurring mobile genetic elements, known as retrotransposons,are also candidates for gene transfer vehicles. This mutagenesis methodgenerally involves the delivery of a gene trap.

Retrotransposons are naturally occurring DNA elements which are found incells from almost all species of animals, plants and bacteria which havebeen examined to date. They are capable of being expressed in cells, canbe reverse transcribed into an extrachromosomal element and reintegrateinto another site in the same genome from which they originated.

Retrotransposons may be grouped into two classes, the retrovirus-likeLTR retrotransposons, and the non-LTR elements such as human L1elements, Neurospora TAD elements (Kinsey, 1990, Genetics 126:317-326),I factors from Drosophila (Bucheton et al., 1984, Cell 38:153-163), andR2Bm from Bombyx mori (Luan et al., 1993, Cell 72: 595-605). These twotypes of retrotransposon are structurally different and alsoretrotranspose using radically different mechanisms.

Unlike the LTR retrotransposons, non-LTR elements (also called polyAelements) lack LTRs and instead end with polyA or A-rich sequences. TheLTR retrotransposition mechanism is relatively well-understood; incontrast, the mechanism of retrotransposition by non-LTRretrotransposons has just begun to be elucidated (Luan and Eickbush,1995, Mol. Cell. Biol. 15:3882-3891; Luan et al., 1993, Cell72:595-605). Non-LTR retrotransposons can be subdivided intosequence-specific and non-sequence-specific types. L1 is of the lattertype being found to be inserted in a scattered manner in all human,mouse and other mammalian chromosomes.

Some human L1 elements (also known as a LINES) can retrotranspose(express, cleave their target site, and reverse transcribe their own RNAusing the cleaved target site as a primer) into new sites in the humangenome, leading to genetic disorders.

Further included in the invention are DNAs which are useful for thegeneration of mutations in a cell. The mutations created are useful forassessing the frequency with which selected cells undergo insertionalmutagenesis for the generation of genetically modified animals and thelike. Engineered L1 elements can also he used as retrotransposonmutagens. Sequences can be introduced into the L1 that increases itsmutagenic potential or facilitates the cloning of the interrupted gene.DNA sequences useful for this application of the invention includemarker DNAs, such as GFP, that are specifically engineered to integrateinto genomic DNA at sites which are near to the endogenous genes of thehost organism. Other potentially useful DNAs for delivery are regulatoryDNA elements, such as promoter sequences, enhancer sequences, retroviralLTR elements and repressors and silencers. In addition, genes which aredevelopmentally regulated are useful in the invention.

Viral Mutagenesis Methods

Viral vectors are often created using a replication defective virusvector with a genome that is partially replaced by the genetic materialof interest (e.g., gene trap, selectable marker, and/or a therapeuticgene). The viral vector is produced by using a helper virus to providesome of the viral components that were deleted in the replicationdefective virus, which results in an infectious recombinant virus whosegenome encodes the genetic material of interest. Viral vectors can beused to introduce an insertion mutation into the rat's genome.Integration of the viral genetic material is often carried out by theviral enzyme integrase. Integrase brings the ends of viral DNA togetherand converts the blunt ends into recessed ends. Integrase createsstaggered ends on chromosomal DNA. The recessed ends of the viral DNAare then joined with the overhangs of genomic DNA, and thesinglestranded regions are repaired by cellular mechanisms. Somerecombinant virus vectors are equipped with cell uptake, endosomalescape, nuclear import, and expression mechanisms allowing the geneticmaterial of interest to be inserted and expressed in the rat's genome.The genetic material introduced via viral vectors can genetically modifythe rat's genome but is not limited to disrupting a gene, inserting agene to be expressed, and by delivery of interfering RNA. Viral vectorscan be used in multiple methods of delivery. The most common mode ofdelivery is the microinjection of a replication deficient viral vector(e.g., retroviral, adenoviral) into an early embryo (1-4 day) or aonecell pronuclear egg. After viral vector delivery, the embryo iscultured in vitro and transferred to recipient rats to creategenetically modified progeny.

In one embodiment, insertion mutations can be created by delivery of agene trap vector into the rat genome. The gene trap vector consists of acassette that contains selectable reporter tags. Upstream from thiscassette is a 3′ splice acceptor sequence. Downstream from the cassettelays a termination sequence poly adenine repeat tail (polyA). The spliceaccepter sequence allows the gene trap vector to be spliced intochromosomal mRNA. The polyA tail signals the premature interruption ofthe transcription. The result is a truncated mRNA molecule that hasdecreased function or is completely non-functional. The gene trap methodcan also be utilized to introduce exogenous DNA into the genome.

In another embodiment an enhancer trap is used for insertionalmutagenesis. An enhancer trap is a transposable element vector thatcarries a weak minimal promoter which controls a reporter gene. When thetransposable element is inserted the promoter drives expression of thereporter gene. The expression of the reporter gene also displays theexpression patterns of endogenous genes. Enhancer trapping results ingenetic modification and can be used for gain-of-function genetics. TheGa14-mediated expression system is an example of an enhancer trap.

Further included are one or more selectable marker genes. Examples ofsuitable prokaryotic marker genes include, but are not limited to, theampicillin resistance gene, the kanamycin resistance gene, the geneencoding resistance to chloramphenicol, the lacZ gene and the like.Examples of suitable eukaryotic marker genes include, but are notlimited to, the hygromycin resistance gene, the green fluorescentprotein (GFP) gene, the neomycin resistance gene, the zeomycin gene,modified cell surface receptors, the extracellular portion of the IgGreceptor, composite markers such as beta-geo (a lac/neo fusion) and thelike.

In one embodiment, the gene trap will need to be integrated into thehost genome and an integrating enzyme is needed. Integrating enzymes canbe any enzyme with integrating capabilities. Such enzymes are well knownin the art and can include but are not limited to transposases,integrases, recombinases, including but not limited to tyrosinesite-specific recombinases and other site-specific recombinases (e.g.,cre), bacteriophage integrases, retrotransposases, and retroviralintegrases.

The integrating enzymes of the present invention can be any enzyme withintegrating capabilities. Such enzymes are well known in the art and caninclude but are not limited to transposases (especially DDEtransposases), integrases, tyrosine site-specific recombinases and othersite-specific recombinases (e.g., cre), bacteriophage integrases,integrons, retrotransposases, retroviral integrases and terminases.

Disclosed are compositions, wherein the integrating enzyme is atransposase. It is understood and herein contemplated that thetransposase of the composition is not limited and to any one transposaseand can be selected from at least the group consisting of SleepingBeauty (SB), Tn7, Tn5, mos1, piggyBac, Himar1, Hermes, Tol2, Pokey,Minos, S elements, P-elements, ICESt1, Quetzal elements, Tn916, maT,Tc1/mariner and Tc3.

Where the integrating enzyme is a transposase, it is understood that thetransposase of the composition is not limited and to any one transposaseand can be selected from at least the group consisting of SleepingBeauty (SR), Tn7, Tn5, Tn916, Tc1/mariner, Minos and S elements, Quetzalelements, Txr elements, maT, mosl, piggyBac, Himar1, Hermes, Tol2,Pokey, P-elements, and Tc3. Additional transposases may be foundthroughout the art, for example, U.S. Pat. No. 6,225,121, U.S. Pat. No.6,218,185 U.S. Pat. No. 5,792,924 U.S. Pat. No. 5,719,055, U.S. PatentApplication No. 20020028513, and U.S. Patent Application No. 20020016975and are herein incorporated by reference in their entirety. Since theapplicable principal of the invention remains the same, the compositionsof the invention can include transposases not yet identified.

Also disclosed are integrating enzymes of the disclosed compositionswherein the enzyme is an integrase. For example, the integrating enzymecan be a bacteriophage integrase. Such integrase can include anybacteriophage integrase and can include but is not limited to lamdabacteriophage and mu bacteriophage, as well as Hong Kong 022 (Cheng Q.,et al. Specificity determinants for bacteriophage Hong Kong 022integrase: analysis of mutants with relaxed core-binding specificities.(2000) Mol Microbiol. 36(2):424-36.), HP1 (Hickman, A. B., et al.(1997). Molecular organization in site-specific recombination: Thecatalytic domain of bacteriophage HP1 integrase at 2.7 A resolution.Cell 89: 227-237), P4 (Shoemaker, N B, et al. (1996). The Bacteroidesmobilizable insertion element, NBU1, integrates into the 3′ end of aLeu-tRNA gene and has an integrase that is a member of the lambdaintegrase family. J Bacteriol. 178(12):3594-600.), P1 (Li Y, and AustinS. (2002) The P1 plasmid in action: time-lapse photomicroscopy revealssome unexpected aspects of plasmid partition. Plasmid. 48(3):174-8.),and T7 (Rezende, L. F., et al. (2002) Essential Amino Acid Residues inthe Single-stranded DNA-binding Protein of Bacteriophage T7.Identification of the Dimer Interface. J. Biol. Chem. 277,50643-50653.). Integrase maintains its activity when fused to otherproteins.

Also disclosed are integrating enzymes of the disclosed compositionswherein the enzyme is a recombinase. For example, the recombinase can bea Cre recombinase, Flp recombinase, HIN recombinase, or any otherrecombinase. Recombinases are well-known in the art. An extensive listof recombinases can be found in Nunes-Duby SE, et al. (1998) Nuc. AcidsRes. 26(2): 391-406, which is incorporated herein in its entirety forits teachings on recombinases and their sequences.

Also disclosed are integrating enzymes of the disclosed compositionswherein the enzyme is a retrotransposase. For example, theretrotransposase can be a GATE retrotransposase (Kogan G L, et al.(2003) The GATE retrotransposon in Drosophila melanogaster: mobility inheterochromatin and aspects of its expression in germ line tissues. MolGenet Genomics. 269(2):234-42).

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination. Thesesystems typically rely on sequence flanking the nucleic acid to beexpressed that has enough homology with a target sequence within thehost cell genome that recombination between the vector nucleic acid andthe target nucleic acid takes place, causing the delivered nucleic acidto be integrated into the host genome. These systems and the methodsnecessary to promote homologous recombination are known to those ofskill in the art.

Zinc Finger Nucleases

In another method, a zinc finger nuclease creates site-specificdeletions via double-stranded DNA breaks that are repaired bynon-homologous end joining (NHEJ). Zinc finger nucleases may also beused to create an insertion mutation by combining the ZFN with ahomologously integrating cassette to create an insertion in the genomicDNA. Therefore, this genetic modification method can be used for bothtargeted (site-specific) DNA insertions and targeted DNA deletions. Inone embodiment, the method involves transformation of a cell with anucleic acid or mRNA construct minimally comprising DNA encoding achimeric zinc finger nuclease (ZFN), which can be used to create a DNAdeletion. In another embodiment, a second DNA construct can be providedthat will serve as a template for repair of the cleavage site byhomologous recombination. In this embodiment, a DNA insertion may becreated. The DNA insertion may contain a gene trap cassette. In oneembodiment, this method can be combined with spermatogonial stem celltechnology or embryonic stem cell technology, as mentioned above. Inanother embodiment, this method can be combined with mobile DNAtechnology. This technique can also be done directly in the rat embryo.

Nucleic Acid Modification Methods

In one embodiment, a random mutation is created with a chemical mutagenand then a screen is performed for insertions in a particular SCID gene.Chemical mutagens such as methane-sulfonic acid ethylester (EMS),N-ethyl-N-nitrosourea (ENU), diepoxyoctane and UV/trimethylpsorlalen maybe employed to create nucleic acid sequence mutations.

Sequence editing methods can also be used that involve the delivery ofsmall DNA fragments, hybrid DNA/RNA molecules, and modified DNA polymersto create sequence mismatches and nucleic acid mutations. RNA/DNAhybrids are molecules composed of a central stretch of DNA flanked byshort RNA sequences that form hairpin structures. The RNA/DNA hybridscan produce single base-pair substitutions and deletions resulting innucleotide mutations. Some other sequence editing examples includetriplex forming oligonucleotides, small fragment homologous replacement,single stranded DNA oligonucleotides, and adeno-associated virus (AAV)vectors.

The invention also is directed to genetic expression modification ormutagenesis by delivery of a transgene that works in trans.

In one genetic modification method, RNA interference may be used toalter the expression of a gene. In another genetic modification method,the delivery of a transgene encoding a dominant negative protein mayalter the expression of a target gene.

Vector Delivery Methods

The mutagenesis methods of this invention may be introduced into one ormore cells using any of a variety of techniques known in the art suchas, but not limited to, microinjection, combining the nucleic acidfragment with lipid vesicles, such as cationic lipid vesicles, particlebombardment, electroporation, DNA condensing reagents (e.g., calciumphosphate, polylysine or polyethyleneimine) or incorporating the nucleicacid fragment into a viral vector and contacting the viral vector withthe cell. Where a viral vector is used, the viral vector can include anyof a variety of viral vectors known in the art including viral vectorsselected from the group consisting of a retroviral vector, an adenovirusvector or an adeno-associated viral vector.

DNA or other genetic material may be delivered through viral andnon-viral vectors. These vectors can carry exogenous DNA that is used togenetically modify the genome of the rat. For example Adenovirus (AdV),Adeno-associated virus (AAV), and Retrovirus (RV) which contain LTRregions flanking a gene trap, transgene, cassette or interfering RNA areused to integrate and deliver the genetic material. Another deliverymethod involves non-viral vectors such as plasmids used forelectroporation and cationic lipids used for lipofection. The non-viralvectors usually are engineered to have mechanisms for cell uptake,endosome escape, nuclear import, and expression. An example would be anon-viral vector containing a specific nuclear localization sequence andsequence homology for recombination in a targeted region of the genome.

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. For example,the nucleic acids can be delivered through a number of direct deliverysystems such as, electroporation, lipofection, calcium phosphateprecipitation, plasmids, cosmids, or via transfer of genetic material incells or carriers such as cationic liposomes. Appropriate means fortransfection, including chemical transfectants, or physico-mechanicalmethods such as electroporation and direct diffusion of DNA, aredescribed by, for example, Wolff, J. A., et al., Science, 247,1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818, (1991). Suchmethods are well known in the art and readily adaptable for use with thecompositions and methods described herein. In certain cases, the methodswill be modified to specifically function with large DNA molecules.Further, these methods can be used to target certain diseases and cellpopulations by using the targeting characteristics of the carrier.

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosednon-viral vectors for example, lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposome, or polymersomes. Liposomes can further comprise proteins tofacilitate targeting a particular cell, if desired. Administration of acomposition comprising a compound and a cationic liposome can beadministered to the blood afferent to a target organ or inhaled into therespiratory tract to target cells of the respiratory tract. Regardingliposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol. Biol.1:95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417(1987); U.S. Pat. No. 4,897,355. Furthermore, the vector can beadministered as a component of a microcapsule that can be targeted tospecific cell types, such as macrophages, or where the diffusion of thecompound or delivery of the compound from the microcapsule is designedfor a specific rate or dosage.

In the methods described above, which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

These vectors may be targeted to a particular cell type via antibodies,receptors, or receptor ligands. The following references are examples ofthe use of this technology to target specific proteins to tumor tissueand are incorporated by reference herein (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battclli, ct al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other specific cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid-mediated drugtargeting to colonic carcinoma), receptor-mediated targeting of DNAthrough cell specific ligands, lymphocyte-directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue and areincorporated by reference herein (Hughes et al., Cancer Research,49:6214-6220, (1989); and Litzinger and Huang, Biochimica et BiophysicaActa, 1104:179-187, (1992)). In general, receptors are involved inpathways of endocytosis, either constitutive or ligand induced. Thesereceptors cluster in clathrin-coated pits, enter the cell viaclathrin-coated vesicles, pass through an acidified endosome in whichthe receptors are sorted, and then either recycle to the cell surface,become stored intracellularly, or are degraded in lysosomes. Theinternalization pathways serve a variety of functions, such as nutrientuptake, removal of activated proteins, clearance of macromolecules,opportunistic entry of viruses and toxins, dissociation and degradationof ligand, and receptor-level regulation. Many receptors follow morethan one intracellular pathway, depending on the cell type, receptorconcentration, type of ligand, ligand valency, and ligand concentration.Molecular and cellular mechanisms of receptor-mediated endocytosis havebeen reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409(1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome typically contain integration sequences. Thesesequences are often viral related sequences, particularly when viralbased systems arc used. These viral integration systems can also beincorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Ada Domains and Loss of Function Mutations

Rattus norvegicus Adenosine deaminase is a 352 amino acid protein.

The protein contains the following active site residues: Leucine (L)-14,Valine (V)-16, Valine (V)-213, Histidine (H)-214, Glycine(G)-216,Glycine (G)-237, Cysteine (C)-269, Serine (S)-294, Aspartic Acid(D)-295-296, and one suspected post-transcriptionally modified site:Alanine (A)-2.

The Ada gene is a 1417 base pair gene with a coding sequence betweenbase pairs 77-1135.

Blackburn et al. (J. Clin. Invest. 112: 332-344, 2003) concluded thatover-expression of Il13 inhibits Ada activity, and increases the amountof adenosine accumulation. Further, adenosine induces Il13 activity inAda−/− mice.

Table: Amino Acid Changes Resulting in SCID

This table displays some amino acid changes that are predicted todisrupt Ada activity.

TABLE Amino Acid changes resulting in SCID B- T- NK- SCID AA change cellcell cell Full Knockout Lys(K)80-Arg(R) − − − Full KnockoutLeu(L)304-Arg(R) − − − Full Knockout Arg(R)101-Trp(W) − − − FullKnockout Arg(R)101-Gln(Q) − − − Full Knockout Arg (R)211-His(H) − − −Full Knockout Val(V)304-Arg(R) − − − Full Knockout Lys(K)80-Arg ® − − −Full Knockout Ala(A)329-Val(V) − − − Full Knockout Leu(L)107-Pro(P) − −− Full Knockout Gly(G)216-Arg(R) − − − Full Knockout Ala(A)329-Val(V) −− − Full Knockout GG-AG transition − − − in intron 10 creates a newsplice acceptor site, and a freamshift mutation Full Knockout G-Atransition at − − − the +1 position of the 5′ splice joint in exon 2Partial Pro(P)297-Gln(Q) Partial Arg(R)76-Trp(W) PartialArg(R)149-Gln(Q) Partial Pro(P)274-Leu(L) Partial Arg(R)211-Cys(C)Partial Ala(A)215-Thr(T) The Ada gene is a 1417 base pair gene with acoding sequence between base pairs 77-1135.

Ada Phenotypes

The Ada gene encodes the protein Adenosine deaminase (Ada) whichcatalyzes the irreversible deamination of adenosine and deoxyadenosinein the purine catabolic pathway. Ada plays an critical role in theregulation of B-, T-, and NK-cell maturation, proliferation, anddifferentiation. Some Ada mutations result in partial loss of functionor “knockdown” and others result in full loss of function mutations or“knockout”.

The Ada activity resulting from a loss of function in one or several Adaeffectors has completely different and variable phenotypes; someresulting in less immunodeficiency and recurrent respiratory infectiondevelopment or no immunodeficiency and respiratory infectiondevelopment. Complete loss of function or “knockout” of Ada resulting inloss of function in all of its effectors always results in early onsetof impaired cellular immunity, and recurrent respiratory and other organinfections progressing to severe organ incompetence in known animalmodels.

TABLE Severe Combined Immunodeficiency Phenotypes IR Chromosomal B T NKSensitivity Location Homozygous phenotype Rag1 Complete − − + None 3q31The thymus will contain fewer loss of cells along with nonerythroidfunction, cells in the spleen leading to Knockout, small lymphoid organsat 3-9 null, weeks. Rag1−/− Thymocytes will be larger than heterozygousand WT rats, and lymphocyte development is interrupted at an immatureage. B- and T-cell differentiation will not occur, and the rats willhave no mature B- and T-cells in lymphoid organs. Rag1 deficient ratswill show no V(D)J rearrangements at age 4-7 weeks. The lack of V(D)Jrecombination being the cause of non-mature B- and T- lymphocytes andSCID phenotype. Only immune system organs, lymphocytes (spleen, thymus,lymph nodes) will lack cells. Other organs will appear normal and bothfemale and male rats will be fertile. Rag2 Complete − − + None 3q31 Thethymus and spleen of loss of Rag2−/− rats will be fewer cells. function,B-cell differentiation and Knockout, maturation will be blocked at anNull, early stage; which will be at or Rag2−/− near the point of Ig geneand onset of VDJ rearrangement. Lack of Ig rearrangments will lead torats that contain no mature B-cells. No mature T-cells will be found asdeveloping thymocyes are arrested at an early stage. No D(J)rearrangements will be detected in Rag2−/− rats. There will be acomplete lack of rearrangement at the TCR locus. Only immune systemorgans, lymphocytes (spleen, thymus, lymph nodes) will lack cells. Otherorgans will appear normal and both female and male rats will be fertile.Ada − − − None 3q42 In Ada−/− rats the thymus and spleen exhibitsgreater than 50% organ-to-body weight reduction. In the thymus, decreasein cortical-medullary demarcation and the Hassel's corpuscles is absent.Spleen, a decreased amount of red blood cells in the red pulp andmegakaryocytes. An 8-fold decrease of lymphoid cells in the thymus; a3-fold decrease in the spleen. In the peripheral circulationlymphophenia with one third of the lymphoid cells in circulation. Theserum displayed a 3-fold decrease of overall immunoglobin. Thymus,increase in Cd4−/Cd8− double negative immature thymocytes and a decreasein double positive and single positive. Spleen, there was a decrease inCd4+ and Cd8+ T- and B- lymphocytes. Dclre1c − − + Yes 17q12.3 Dclre1cdeficient rats will show defects in opening and joining V(D)J-codinghairpin ends and increased cellular ionizing radiation sensitivity.Nhej1 − − + Yes, with 9q33 Nhej1 deficient rats will have microcephalyimpairments in ability to form and both V(D)J coding joins and growthjoins of their flanking retardation recombination signal sequences. Theywill be highly sensitive to ionizing radiation and have DNA Doublestrand break (DSB) repair defects. Jak3 + − − None 16p14 Jak3 deficientrats will show severe B-cell development defects at the pre-B stage.T-cell development will progress normally, but in response to mitogenicsignals T-cells will not proliferate and will secrete small amounts ofIl-2. Il7r + − + None 2q16 Il7 deficient rats will have a significantreduction in thymic and peripheral lymphoid cellularity. Ptprc + − +13813 Fever, rash, (Cd45) hepatosplenomegaly, lymphadenopathy,pneumonitis, pancytopenia, and cytomegalovirus infection. Low T-cellcount, decreased immunoglobulins, and abnormal Cd45, Cd3d, Cd3eexpression Cd3d + − + 8q22 Cd3d−/− rats exhibit fever, rash,hepatosplenomegaly, lymphadenopathy, pneumonitis, pancytopenia, andcytomegalovirus infection. Low T-cell count, decreased immunoglobulins,and abnormal Cd45, Cd3d, Cd3e expression Cd3e + − + 8q22 Cd3e−/− ratsexhibit fever, rash, hepatosplenomegaly, lymphadenopathy, pneumonitis,pancytopenia, and cytomegalovirus infection. Low T-cell count, decreasedimmunoglobulins, and abnormal Cd45, Cd3d, Cd3e expression Il2rg + − −Yes Xq31 Rats deficient in Il2rg will have the absolute number oflymphocytes reduced to around 10-fold of WT. Prkdc − − + None 11q23Prkdc deficient rats will exhibit lymphopenia, hypogammaglobulinemia,and impaired immune functions mediated by T- and B- lymphocytes.

CLUSTAL 2.0.10 multiple sequence alignment of rat and mouse AdenosineDeaminase (Ada) amino acid sequence. The sequence alignment shows closehomology between the mouse and rat Ada sequence. The homology ofconserved domains and knowledge of insertion mutagenesis allows evidencethat mutagenesis will create a total knockout rat Ada.

Rattus MAQTPAFNKPKVELHVHLDGAIKPETILYYGKKRGIDLPADTVEGLRNIIGMDKPLSLPD  60Mus MAQTPAENKPKVELHVHLDGAIKPETILYEGKKRGIALPADTVEELRNIIGMDKPLSLPG  60*****************************:****** ******* **************. RattusFLAKFDYYMPAIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPIPWNQA 120 MusFLAKFDYYMPVIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQT 120**********.*******************************************:****: RattusEGDLTPDEVVDEVNQGLQEGEQAFGIKVRSILCCMRHQPSWSPEVLELCKKYHQKTVVAM 180 MusEGDVTPDDVVDLVNQGLQEGEQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAM 180***:***:********************************** *********:******* RattusDLAGDETIEGSSLFPGHVEAYEGAVKDGIHRTVHAGEVGSAEVVREAVDILKTERVGHGY 240 MusDLAUDEITEGSSLFPGHVEAYEGAVKNGIHRTVHAGEVGSPEVVREAVDILKIERVGHGY 240**************************:*************:******************* RattusHTIEDEALYNRLLKENMHFEVCPWSSYLTGAWNPKTTHAVVRFKDDQANYSLNSDDPLIF 300 MusHTIEDEALYNRLLKENMHFEVCPWSSYLTGAWDPKTTHAVVRFKNDKANYSLNTDDPLIF 300********************************:***********:*:******:****** RattusKSTVDTDYQMVKKDMGFTEEEFKRLNINAAKSSFLPEDEKKELLERLYKEYQ 352 MusKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKELLERLYREYQ 352***:******.**************************:**********:***

Adenosine Deaminase (Ada−/−) Knockout, complete loss of functionphenotypes

Blackburn et al. (PNAS 92: 9, 3673-7, 1995) created Ada −/− KO mice byintroducing an Ada gene trap which contained the neomycin resistance andherpes simplex tk genes for selection. The mutation resulted in a nullAda allele. The thymus and spleen of Ada −/− KO mice exhibited greaterthan 50% organ-to-body weight reduction. In the thymus, there was adecrease in cortical-medullary demarcation and the Hassel's corpuscleswere absent. In the spleen, a decreased amount of red blood cells in thered pulp and a minuscule amount of megakaryocytes were present. Therewas an 8-fold decrease of lymphoid cells in the thymus, and a 3-folddecrease in the spleen. In the peripheral circulation, lymphopheniaexisted with only one-third of the normal number of lymphoid cells inthe circulation. The serum displayed a more than 3-fold decrease ofoverall immunoglobin. In the thymus, there was a significant increase inCd4-/ Cd8-double negative immature thymocytes and a decrease in doublepositive and single positive thymocytes. In the spleen there was adecrease in Cd4+ and Cd8+T- and B- lymphocytes. T- and B-celllymphopenia and hypogammaglobulinemia were consequences of Adadeficiency. These data, along with the sudden and severe decrease inimmune system competency and health, leading to death (around an age of3 weeks), indicates that the Adenosine Deaminase (Ada-−) KO deficiencyis a severe combined immunodeficiency (SCID) phenotype.

Adenosine Deaminase (Ada) knockdown, partial loss of function phenotype.

Blackburn et al. (J Biol Chem.273: 9:5093-100, 1998) created partialAda-deficient mice by intercrossing male mice which were homozygous forthe null Ada allele with female mice which were heterozygous for thenull Ada allele. These partial Ada-deficient mice only expressed Ada inthe gastrointestinal organs. The partial Ada−/− mice exhibit a muchslower progression of symptoms than the complete Ada−/− KO mice. At 6weeks of age, the lungs accumulated macrophages in the alveolar spaces.At 10 weeks lungs of partial Ada−/− mice exhibited severe inflammation,activated alveolar macrophages, perivascular and peribrochialaccumulation of leukocytes and alveolitis. At 12 weeks of age, the lungsdemonstrated capacious bronchial plugging. Further, the partial Ada−/−mice have an increased level of lung Adenosine that increases with ageand progression of phenotype. These data, demonstrate that the partialAda−/− mice phenotype is one of slower progression and less pronouncedimmunodeficiency than the complete Ada−/− knockout phenotype.

Conditional SCID Phenotype

Mutations in Ada are responsible for ˜20% of the cases of SCID inhumans. Human patients have small or absent thymuses and severelydepressed populations of T-, B-, and NK-cells (<10% normal levels).Renal, pulmonary, and liver abnormalities are observed as well. AdaKnockout (KO) mice exhibit lethality due to defects unrelated to theimmunodeficiency, namely, severe liver dysfunction. Ada KO rats exhibitsimilar liver defects leading to early death. It would be preferable tomodulate the severe liver defect to facilitate the use of rat Ada KOsfor long term propagation of human xenografts for drug discovery ortherapeutic studies.

Two strategies can be used to modulate the severity of non-SCIDphenotypes:

First, it was found that transgenic mice that express Ada under thetranscriptional control of a trophoblast-specific promoter provide ADAactivity only during gestation and that severity of the liver defectswas modulated. Analysis of the peripheral blood from 15-17 day Ada KOneonates showed that these animals still retained the SCID phenotype.

In SCID patients and in Ada KO mice, injection of ADA enzyme coupled topolyethylene glycol for stabilization (PEG-ADA) was used to treatpatients (or KO animals). Interestingly, Ada KO mice exhibitdose-dependent responses to PEG-ADA treatment: at low doses, Ada KO miceare protected from the early lethal defects, such as the severe liverdefects, but still retained the SCID phenotype.

The PEG-ADA dosage experiments indicate that any dose-dependentconditional transgenic system that can produce defined levels of ADAenzyme in the ADA (SCID) mutant background would similarly modulate thenon-immune defects, and thus create SCID models that would be amenablefor long term propagation of human xenografts. An example of such asystem, but not limited to this example, are doxycycline-induciblesystems (so called tet-on or tet-off systems) which can be used toconditionally complement the non-immune defects, while still maintainingthe SCID phenotype.

EXAMPLES

The rat and progenies thereof of the present invention may be any rat orprogenies thereof, so long as they are a rat or progenies thereof inwhich genome is modified so as to have decreased or deleted activity ofthe severe combined immunodeficiency (SCID) gene.

Gene Disruption Technique which Targets at a Gene Encoding AdenosineDeaminse (Ada)

The gene disruption method may be any method, so long as it can disruptthe gene of the target enzyme. Examples include a homologousrecombination method, a method using retrovirus, a method using DNAtransposon, and the like.

(a) Preparation of the rat and progenies thereof of the presentinvention by homologous recombination

The rat and the progenies thereof of the present invention can beproduced by modifying a target gene on chromosome through a homologousrecombination technique which targets at a gene encoding the SCID gene.The target gene on chromosome can be modified by using a methoddescribed in Gene Targeting, A Practical Approach, IRL Press at OxfordUniversity Press (1993) (hereinafter referred to as “Gene Targeting, APractical Approach”); or the like, for example.

Based on the nucleotide sequence of the genomic DNA, a target vector isprepared for homologous recombination of a target gene to be modified(e.g., structural gene of the SCID gene, or a promoter gene). Theprepared target vector is introduced into an embryonic stem cell and acell in which homologous recombination occurred between the target geneand target vector is selected.

The selected embryonic stem cell is introduced into a fertilized eggaccording to a known injection chimera method or aggregation chimeramethod, and the embryonic stem cell-introduced fertilized egg istransplanted into an oviduct or uterus of a pseudopregnant female rat tothereby select germ line chimeras.

The selected germ line chimeras are crossed, and individuals having achromosome into which the introduced target vector is integrated byhomologous recombination with a gene region on the genome which encodesthe SCID protein are selected from the born offsprings.

The selected individuals are crossed, and homozygotes having achromosome into which the introduced target vector is integrated byhomologous recombination with a gene region on the genome which encodesthe SCID protein in both homologous chromosomes are selected from theborn offsprings. The obtained homozygotes are crossed to obtainoffspring to thereby prepare the rat and progenies thereof of thepresent invention.

(b) Preparation of the rat and progenies thereof of the presentinvention by a method using a transposon

The rat and progenies thereof of the present invention can be preparedby using a transposon system similar to that described in Nature Genet.,25, 35 (2000) or the like, and then by selecting a mutant of the SCIDgene.

The transposon system is a system in which a mutation is induced byrandomly inserting an exogenous gene into chromosome, wherein an genetrap cassette or exogenous gene interposed between transposons isgenerally used as a vector for inducing a mutation, and a transposaseexpression vector for randomly inserting the gene into chromosome isintroduced into the cell at the same time. Any transposase can be used,so long as it is suitable for the sequence of the transposon to be used.As the gene trap cassette or exogenous gene, any gene can be used, solong as it can induce a mutation in the DNA of the cell.

The rat and progenies thereof of the present invention can be preparedby introducing a mutation into a gene encoding the severe combinedimmunodeficiency (SCID) associated protein, and then by selecting a ratof interest in which the DNA is mutated.

Specifically, the method includes a method in which a rat of interest inwhich the mutation occurred in the gene encoding the Ada protein isselected from mutants born from generative cells which are subjected tomutation-inducing treatment or spontaneously generated mutants. Inanother embodiment, the SCID gene is one of several known SCID genes,such as (Rag1, Rag2, Dclre1c, Nhej1, Jak3, Il 7r, Ptprc, Cd3d, Cd3e,Il2rg, Prkdc, Sirpa, Foxn1). The generative cell includes cells capableof forming an individual such as a sperm, an ovum or a pluripotentcells. The generative cell may also be a somatic cell and the animal maythen be created by somatic cell nuclear transfer.

Examples in which several methods described above have been employed bythe inventors to create a SCID model phenotype in Rattus norvegicus aredescribed below.

Genetic modification to Rattus norvegicus SCID gene Adenosine Deaminase(Ada) was carried out by a DNA transposon insertional mutagenesis methodsimilar to that described in Nature Genet., 25, 35 (2000). The DNAtransposon-mediated genetically modified allele was designatedAdaTn(sb-T2/Bart3)2.237Mcwi. The mutant strain symbol for the SCID ratwas designated F344-AdaTn(sbT2/Bart3)2.237Mcwi.

The DNA transposon insertion occurred in chromosome 3, within intron 7of the rat Ada gene. The sequence tag map position was between basepairs: 154638511-154638627. The sequence tag was:TAGGTTCCTGGGTTCAAACTCAGGTTGTCATGCTTTGTGGAAGGCACCTTCACCCACTGAGCCATCTTACCAGTTCCAGAATTTGACACTTGACTTTTCTCAAAGCACTATTCCTAG.

Thus, a DNA transposon was inserted into the Ada gene of Rattusnorvegicus rendering the gene completely inactive. Adenosine Deaminase(Ada −/−) KO rats have no mature B-, T-, or NK cells. There was anaccumulation of 2-deoxyadenosine and dATP, and a decreased level ofS-adenosylhomocysteine hydrolase in immune organs. The phenotype wasthat of a Severe Combined Immunodeficient (SCID) rat.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology and biochemistry,which are within the skill of the art.

1.-53. (canceled)
 54. A genetically modified non-human mammal, orprogenies thereof, at least some of whose cells comprise a genomecomprising a genetic mutation in one or more genes that causes themammal to have a greater susceptibility to Severe CombinedImmunodeficiency (SCID) than a mammal not comprising the geneticmutation.
 55. The mammal of claim 54, wherein said gene is selected fromthe group consisting of Ada, Rag1, Rag2, Dclre1c, Nhej1, Jak3, Il7r,Ptprc, Cd3d, Cd3e, Il2rg, Prkdc, Sirpa, Foxn1, genes having at leastabout 80% sequence identity to a gene selected from Ada, Rag1, Rag2,Dclre1c, Nhej1, Jak3, Il7r, Ptprc, Cd3d, Cd3e, Il2rg, Prkdc, Sirpa,Foxn1, and combinations thereof.
 56. The genetically modified nonhumanmammal of claim 54, wherein the mammal is a rat.
 57. The geneticallymodified nonhuman mammal of claim 54, wherein one or more SCID genes orloci are misexpressed and/or conditionally misexpressed.
 58. Thenon-human animal model of claim 54, wherein the genetic mutation resultsin decreased expression of one or more SCID proteins.
 59. Thegenetically modified nonhuman mammal of claim 54, wherein the one ormore genes encoding a SCID protein is disrupted.
 60. The geneticallymodified nonhuman mammal of claim 54, wherein the SCID gene is selectedfrom the group consisting of Ada, Rag1, Rag2, Jak3, and Prkdc.
 61. Thegenetically modified nonhuman mammal of claim 57, wherein the cells arepluripotent cells.
 62. The genetically modified nonhuman mammal of claim54, wherein the one or more SCID genes or loci are disrupted by amechanism selected from mutating directly in the germ cells of a livingorganism; removal of DNA encoding all or part of the SCID protein;insertion mutation; transposon insertion mutation; deletion mutation;introduction of a cassette or gene trap by recombination; chemicalmutagenesis; RNA interference (RNAi); mutagenesis by site-specificnuclease; or delivery of a transgene encoding a dominant negativeprotein, which may alter the expression of a target gene.
 63. Thegenetically modified nonhuman mammal of claim 54, wherein the one ormore SCID genes or loci are disrupted by a transposon insertionmutation.
 64. The genetically modified nonhuman mammal of claim 61,wherein the disruption causes a complete or partial loss-of-functionphenotype.
 65. The genetically modified nonhuman mammal of claim 54,wherein said gene is capable of being regulated via transcriptionalcontrol.
 66. The genetically modified nonhuman mammal of claim 54,wherein said genetic mutation is conditionally complemented byconditional expression of a transgene.
 67. A screening method foridentifying useful compounds, comprising (a); contacting a rat modelsystem comprising a genetically modified nonhuman mammal of claim 54, orprogenies thereof, at least some of whose cells comprise a genomecomprising a genetic mutation in one or more SCID genes that causes themammal to have a greater susceptibility to Severe CombinedImmunodeficiency (SCID) induction than a mammal not comprising thegenetic mutationwith a candidate test agent; and (b) detecting aphenotypic change in the model system that indicates that the SCIDfunction is restored when compared relative to wild-type cells.
 68. Thescreening method of claim 67, wherein the method is used for identifyinguseful compounds for the treatment of a disease or condition selectedfrom the group consisting of hyperproliferative disorders,transplantation conditions, stem cell disorders, and immunologicaldisorders.